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Neuroprotective Effects of Dantrolene in
Neurodegenerative Disease: Role of Inhibition of
Pathological Inflammation
Wenjia Zhang, Xu Zhao, Piplu Bhuiyan, Henry
Liu, Huafeng Wei
PII: S2957-3912(24)00057-3
DOI: https://doi.org/10.1016/j.jatmed.2024.04.002
Reference: JATMED7
To appear in: Journal of Anesthesia and Translational Medicine
Please cite this article as: Wenjia Zhang, Xu Zhao, Piplu Bhuiyan, Henry Liu and
Huafeng Wei, Neuroprotective Effects of Dantrolene in Neurodegenerative
Disease: Role of Inhibition of Pathological Inflammation, Journal of Anesthesia
and Translational Medicine, (2024)
doi:https://doi.org/10.1016/j.jatmed.2024.04.002
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© 2024 Published by Elsevier.
Neuroprotective Effects of Dantrolene in Neurodegenerative Disease:
Role of Inhibition of Pathological Inflammation
Wenjia Zhang1,2, Xu Zhao 2, Piplu Bhuiyan 1,
Henry Liu 1, Huafeng Wei 1
1Department of Anesthesiology and Critical Care
Perelman School of Medicine
University of Pennsylvania
Philadelphia, PA 19104, USA
2Department of Anesthesiology
Shandong Provincial Hospital
Shandong First Medical University
Jinan, Shandong, 250021, China
Corresponding Author:
Huafeng Wei, MD, PhD
Department of Anesthesiology and Critical Care
University of Pennsylvania Perelman School of Medicine
305 John Morgan Building
3620 Hamilton Walk
Philadelphia, PA 19104
Phone: 215-2609840, FAX: 215-349-507
Email: huafeng.wei@pennmedicine.upenn.edu
Abstract
Neurodegenerative diseases (NDs) refer to a group of diseases in which slow,
continuous cell death is the main pathogenic event in the nervous system. Most
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NDs are characterized by cognitive dysfunction or progressive motor
dysfunction. Treatments of NDs mainly target alleviating symptoms, and most NDs do
not have disease-modifying drugs. The pathogenesis of NDs involves inflammation
and apoptosis mediated by mitochondrial dysfunction. Dantrolene, approved by
the US Food and Drug Administration, acts as a RyRs antagonist for the
treatment of malignant hyperthermia, spasticity, neuroleptic syndrome, ecstasy
intoxication and exertional heat stroke with tolerable side effects. Recently,
dantrolene has also shown therapeutic effects in some NDs. Its neuroprotective
mechanisms include the reduction of excitotoxicity, apoptosis and
neuroinflammation. In summary, dantrolene can be considered as a potential
therapeutic candidate for NDs.
Keywords:
Alzheimer's disease, cognitive dysfunction, progressive motor dysfunction,
apoptosis, calcium, pyroptosis
Introduction
Neurodegenerative diseases (NDs) refer to a group of diseases
characterized by progressive and chronic cell death within the nervous system,
manifesting clinically as symptoms reflective of the pathophysiological changes
caused by neuronal loss [1]. Many diseases can be classified into NDs. The
prototypical examples include Parkinson's disease (PD), Alzheimer's disease
(AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and
multiple sclerosis (MS). Aging is the biggest risk factor for most NDs [2].
Common NDs and their estimated prevalence, as reported in primary literature,
are shown in Table 1.
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Table 1 Prevalence and major symptoms of common NDs
Disease
Prevalence
Major Symptoms
Alzheimer's
disease
(AD)
6.7 million age 65 and
older in the US in 2023[2]
Impairment of learning and
memory, speech difficulties
Parkinson's
disease
(PD)
2–3% of the global
population aged
>65 years in 2017 [3]
Muscle tremor, rigidity,
bradykinesia/akinesia, and
postural instability
Huntington's
disease
(HD)
10.6–13.7 per 100 000 in
Western countries. 1–7 per
million in Japan, Taiwan,
and Hong Kong in 2017 [4]
Chorea, dystonia, loss of
coordination, cognitive
decline,
behavioral difficulties
Amyotrophic
lateral
sclerosis
(ALS)
Prevalence rates (per
100,000 persons) and
incidence rates (per
100,000 person-years) are
6.22 and 2.31 for Europe,
5.20 and 2.35 for North
America, 3.41 and 1.25 for
Latin America, 3.01 and
0.93 for Asian countries
excluding Japan, and 7.96
and 1.76 for Japan,
respectively.[5]
Progressive motor defects,
with
muscle weakness, atrophy
and
spasms
Multiple
sclerosis
(MS)
2.8 million people
worldwide in 2021[6]
Blurred vision, weak limbs,
tingling sensations,
unsteadiness, and fatigue
Most NDs are characterized by cognitive dysfunction or progressive motor
dysfunction, thus placing an increasing burden on caregivers and economy.
More importantly, PD stands as the the only treatable ND currently with the
individualized treatment objectives, emphasizing the need of personalized
management. Levodopa serves as the primary first-line treatment. Achieving
optimal treatment begins with diagnosis and requires a multidisciplinary
approach that includes a number of nonpharmacological interventions [7]. All
other NDs are incurable. To date, two types of drugs have received approval
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for AD, including cholinesterase inhibitors and the N - methyl - D - aspartate
(NMDA) receptor antagonist. However, these medications only alleviate the
symptoms of AD and do not provide a cure or preventive measures against the
disease itself. [8]. Potential interventions for HD include therapies targeting
huntingtin DNA and RNA, clearance of huntingtin protein, DNA repair pathways,
and other treatment strategies targeting inflammation and cell death [9]. For
ALS, survival can be improved through supportive and symptomatic care
delivered by a multidisciplinary team of experts. This approach may involve
expert consensus guidelines and an evidence-based approach to treatment.
[10]. The greatest challenge in MS lies in developing therapies that combine
neuroprotection and remyelination to treat, and ultimately disable, the
progression of the disease [11]. Currently, many disease-modifying therapies
for patients in early aggressive courses have shown variable efficacy in
preventing recurrence, lesion accumulation on magnetic resonance imaging
(MRI), and disability progression [12].
Dantrolene, a Food and Drug Administration (FDA)-approved drug for the
treatment of malignant hyperthermia, has recently been evaluated for
prospective use as a neuroprotective agent for the treatment of
neurodegenerative syndromes including AD [13]. Researchers have
demonstrated that dantrolene improves memory loss and neuropathology in
different animal models of AD [14, 15]. Through in-vitro experiments,
researchers have also demonstrated that dantrolene ameliorated the
impairments in neurogenesis and synaptogenesis [16]. Recent studies also
show dantrolene’s therapeutic effects in different NDs including AD [14, 15] and
HD [80]. The potential mechanisms by which dantrolene protects neurons are
decreasing excitotoxicity, apoptosis, and neuroinflammation.
Pathogenesis of NDs and potential therapeutic pathways
Although NDs are heterogeneous and contain a series of different diseases,
the study of genetic defects and disease-related proteins indicates that different
NDs share similar features, mechanisms, and even genetic or protein
abnormalities [17].
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There are two main mechanisms that may explain this challenging process.
One is an inflammation-mediated immune response, and the other is apoptosis,
a form of programmed cell death. The two mechanisms are implicated in the
pathogenesis of NDs, including AD, PD, HD, MS, and ALS [18, 19].
To date, mitochondrial dysfunction has been thought to contribute to
inflammation and apoptosis in NDs through the accumulation of mitochondrial
DNA (mtDNA) mutations and the generation of reactive oxygen species (ROS)
[17, 20]. Specific interactions of disease-related proteins with mitochondria
have recently been uncovered: APP, β-amyloid (Aβ), presenilin, α-synuclein,
parkin, DJ-1, PINK1 (phosphatase and tension homologue-induced kinase 1),
LRRK2 (leucine-rich-repeat kinase 2), HTRA2, SOD1 and huntingtin have all
been demonstrated to localize within the mitochondria [20] [21].
The pathology of inflammation and mitochondrial dysfunction-mediated
apoptosis in AD is characterized by the accumulation of amyloid β-containing
neurotic plaques, neurofibrillary tangles, and dystrophic neurites containing
hyperphosphorylated tau protein. Microglia, the resident macrophages in the
brain, become activated by pathological events such as neuronal death or
protein aggregates. Once activated, they can bind to Aβ oligomers and Aβ fibrils
via cell surface receptors. SCARA1, CD36, CD14, α6β1 integrin, CD47, and
Toll-like receptors (TLR2, TLR4, TLR6, and TLR9) are receptors. The process
of microglia activation is thought to be part of the inflammatory reaction in AD
[22]. Inefficient clearance of Aβ has been identified as a major pathogenic
pathway in sporadic cases of AD [22]. By decreasing the expression of Aβ
phagocytosis receptors, microglia show insufficient phagocytic capacity which
is thought to be responsible for increased cytokine concentration. Reduced
mitophagy process enhances neuroinflammation due to reduced phagocytosis
and over stimulation of microglia, leading to increased accumulation of Aβ
plaques and hyperphosphorylated tau protein [22, 23]. In addition, the
researchers showed that in TgAPPsw and PSAPP transgenic mice, the
increases of Aβ concentration were highly correlated with increased
concentration of proinflammatory cytokines, including TNFα, interleukin 6 (IL-
6), interleukin 1α (IL-1α), and granulocyte-macrophage colony stimulating
factor (GM-CSF) [22].
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Oxidative damage in the brain of AD transgenic APP mice proceeds the
occurrence of Aβ deposition and significant plaque pathological changes. The
signaling pathways of APP or tau processing may be activated by oxidative
stress. In vitro, studies have shown that exposure to Aβ increases oxidative
stress and decreases energy availability, thereby increasing the susceptibility
of cell death by inducing apoptosis. However, evidence regarding the role of
apoptosis in neuronal cell death in AD remains limited [24, 25].
Inflammation and mitochondrial dysfunction-mediated apoptosis are
important pathologies of PD. The pathological feature of PD is the presence of
Lewy bodies, resulting from α-synuclein aggregation and dopaminergic
neurodegeneration in the substantia nigra pars compacta. Dopaminergic
neurons are selectively vulnerable to mitochondrial dysfunction. Subsequently,
the mitochondrial PT pore opens, resulting in the release of cytochrome c into
the cytoplasm, triggering the mitochondria-mediated apoptotic pathway, which
is considered to be the primary mechanism of dopaminergic neuronal death in
PD. MPTP (1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine), whose metabolite
MPP+ inhibits complex I of the mitochondrial electron-transport chain, causing
parkinsonism in designer-drug abusers [26, 27].
Another important pathogenesis is pro-inflammatory immune-mediated
mechanisms. Results from animal models confirmed the activation of microglia
and the upregulation of pro-inflammatory mediators such as cytokines found in
postmortem PD brains [18]. Antigens such as infectious pathogens, prions,
pathologically modified proteins, aggregates, and apoptotic cells can activate
microglia. Activated microglia subsequently secrete a wide range of
inflammatory mediators, such as TNFα, IL-6, nitric oxide synthase 2 (NOS2),
COX2, and ROS, leading to cell proliferation, followed by slow degeneration,
and finally the death of dopaminergic neurons [28]. Furthermore, clinical studies
in PD patients examined the CSF and peripheral blood, showing elevated
serum IL-1, IL-6, and TGF-β in CSF [29]. Recently, mitochondrial dysfunction
has been proven to induce neuroinflammation [18].
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Inflammation and mitochondrial dysfunction also mediate apoptosis in HD.
HD is caused by mutations in an abnormally expanded CAG trinucleotide
repeat in a gene encoding the polyglutamine repeats in the huntingtin protein.
Inflammation in HD can be triggered by glial cells responses to autonomous
neuronal degeneration or immune cells activation due to mHtt (mutant HTT
protein) expression, or both at the same time [30]. The main inflammatory
processes are carried out by activated microglia and astrocytes. Reactive
astrocytes are observed in presymptomatic HD and correlate with disease
severity [31].
Studies have shown that the activity of complexes II and III of the electron-
transport chain is reduced in the brains of HD patients. Additionally,
mitochondrial respiration and ATP production were also significantly impaired
in striatal cells in mutant HTT-knock-in mouse embryos. Moreover, cytochrome
c discharge, BAX overexpression, and active caspase-3, -8, and -9 have been
illustrated in the brains of HD patients and in experimental models of HD [32].
These evidence suggest that mitochondrial dysfunction-mediated apoptosis
plays an important role in the pathogenesis of HD.
Inflammation and mitochondrial dysfunction mediate apoptosis in ALS.
ALS is recognized by the progressive and specific loss of motor neurons in the
cortex and the ventral horn of the spinal cord. Studies in ALS patients and
animal models have shown that activated microglia and astroglia,
proinflammatory peripheral lymphocytes, and macrophages are associated
with ALS [33]. The anti-inflammatory response is initially protective, but
subsequently, inflammatory cytotoxic cytokines (IL-1β, IL-6, IL-18, TNF-α etc.)
and reactive oxygen species (ROS) can lead to cell death and further tissue
damage [34]. In recent years, an increasing number of studies have revealed
that both CNS and peripheral immune cells release extracellular vesicles, which
can regulate the behavior of neighboring receptor cells and play an essential
role in the pathogenesis of ALS [33].
The spinal cord, nerve, and muscle biopsy samples showed abnormal
mitochondria structures and numbers. The study focused on the expression of
mutant SOD1 in animal and cellular models of ALS. In fact, SOD1 plays a vital
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role in the clearance of ROS, and the abnormal activity of mutant human SOD1
(mSOD1) leads to oxidative damage [35]. Transgenic mice overexpressing the
G93A Sod1 mutation exhibit impaired mitochondrial energy metabolism in the
brain and spinal cord during disease onset. The illustration of DNA
fragmentation has suggested the role of apoptosis in ALS pathogenesis,
Caspase 9 activation, cytochrome c discharge, BAX overexpression, and
reduced Bcl-2 expression in postmortem tissue and transgenic mouse models
of ALS[36-38].
Inflammation and mitochondrial dysfunction mediated apoptosis in MS.
The pathological hallmark of all MS phenotypes is focal plaques (also called
lesions), which are areas of demyelination typically located around post-
capillary venules and characterized by breakdown of the blood-brain barrier
(BBB). The mechanisms underlying BBB breakdown are not fully understood
but seem to involve direct action of pro-inflammatory cytokines and chemokines
(such as TNF-α, IL-1β ,and IL-6) produced by resident and endothelial cells, as
well as indirect cytokine-dependent and chemokine-dependent leukocyte-
mediated damage [39]. BBB dysregulation increases the trans-endothelial
migration of activated leukocytes, including macrophages, T cells, and B cells,
into the CNS. This process leads to further inflammation and demyelination,
resulting in oligodendrocyte loss, reactive gliosis, and neuro-axonal
degeneration[39].
In addition to inflammatory mechanism, researchers are currently focusing
on the role of mitochondrial damage in demyelination and neurodegeneration.
Biochemical studies identified impaired NADH dehydrogenase activity and
increased complex IV activity in mitochondria in MS lesions[40]. Furthermore,
in oligodendrocytes, mitochondrial damage results in the release of apoptosis-
inducing factor, its translocation into the nuclei, and the activation of poly-ADP-
ribose polymerase (PARP), a mechanism demonstrated in oligodendrocyte
destruction and demyelination induced by cuprizone intoxication in an
experiment in vivo [40].
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Therapeutic pathways
Given the growing evidence supporting the role of inflammation and
mitochondrial dysfunction-mediated apoptosis in the pathogenesis of various
NDs, treatments involving anti-inflammation and improving mitochondrial
function may serve as potential therapeutic approaches.
1. Immunomodulatory therapies
Experimental evidence and animal studies showed that non-steroidal anti-
inflammatory drugs (NSAIDs), particularly ibuprofen and piroxicam have
promising results and appeared to reduce PD risk [18]. Other potential
immunomodulatory therapies, such as anti-TNF therapies, are based on in vitro
studies and need more in vivo evidence[18]. Drugs such as PPARγ agonists,
COX inhibitors, vitamin E, vitamin C, curcumin and catechin, which reduce the
neuroinflammatory pathways, are thought to prevent the progression of AD[23].
Preclinical studies in HD mouse models have shown that active immunity is
achieved by the introduction of an immunogenic short peptide. This peptide
generates host B cell-mediated antigen-specific antibodies against exon1 of
mutant HTT protein. Consequently, this approach downregulates aberrant
neuroinflammatory and cell death pathways[31]. Minocycline, which can
suppress microglial activation and modulate apoptosis, was shown to reduce
motor neurons loss, delay disease onset, and extend the survival of SOD1G93A
mice. NP001, which has been shown to be safe and well tolerated, can slow
disease progression by modulating monocyte activation and downregulating
NF-kB in macrophages[41].
2. Inhibition of mitochondrial cytochrome c release
Minocycline can also inhibit mitochondrial cytochrome c release, which is
an essential step in the progression of the mitochondria-mediated apoptotic
pathway. Moreover, it displays broad neuroprotection in experimental models
of NDs[42, 43]
3. The use of antioxidants
One of the most studied antioxidant compounds is mitoquinone (MitoQ),
which exerts direct antioxidant action by scavenging superoxide, peroxyl, and
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peroxynitrite ROS[44]. In an AD transgenic mouse model expressing three
human mutant genes, APP, PSEN1 and tau, MitoQ treatment showed improved
behavioral phenotype. Other antioxidants, such as Skulachev (SkQ1), MitoApo,
melatonin, have also been reported to show neuroprotective effects in different
research models [45-48].
4. Overexpression of the anti-apoptotic protein Bcl-2
CGP 3466 is a drug that ultimately results in the upregulation of anti-
apoptotic Bcl-2. CGP 3466 has been reported to rescue dopaminergic neurons
from death and to subsequently inhibit the development of motor symptoms in
rodent models of PD by preventing mitochondria-mediated apoptosis[49].
Additionally, Bcl-2 overexpression and the deletion of pro-apoptotic BAX and
BAK have been shown to exert neuroprotection in ALS mice by blocking the
apoptosis of lumbar spinal motor neurons and delaying the onset of
symptoms[50].
Dantrolene Pharmacology
Dantrolene is the only effective treatment for malignant hyperthermia (MH),
a fatal condition during general anesthesia. Before the advent of dantrolene,
the mortality rate of MH was reported as high as 80%[51]. Dantrolene was
approved by the Food and Drug Administration (FDA) in 1979, and can
effectively reverse the symptoms and reduce the mortality to less than 5%[52].
Mechanism of action
The ryanodine receptors (RyRs) are major intracellular Ca 2+ release
channels located in the plasma membrane of the endoplasmic/sarcoplasmic
reticulum. Three mammalian isoforms of RyRs have been isolated. Type 1
RyRs (RyR-1) is preferentially expressed in skeletal muscle and in cerebellar
Purkinje neurons. Type 2 RyRs (RyR-2) is predominantly expressed in cardiac
muscle and is the most abundant isoform in the brain. Type 3 RyRs (RyR-3)
was first identified in the brain and is mainly found in cortical and hippocampal
regions involved in learning and memory[53]. Both RyR-1 and RyR-3 are the
targets of dantrolene in vivo, while previously RYR2 in cardiac SR vesicles was
thought insensitive to clinical concentrations of dantrolene[54]. A recent in vitro
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study shows that the inhibition of dantrolene to RyR2 complex is very sensitive
but depends on the presence of both FKBP12.6(a regulatory protein) and
calmodulin (CaM)[55].
The hypermetabolic symptoms of MH are due to the abnormal release of
the Ca2+ from the sarcoplasmic reticulum (SR) via RyR-1 in skeletal
muscles[56]. Symptoms of chronic ischemia heart disease including ventricular
tachycardia, left-ventricular remodeling, and contractile dysfunction are highly
associated with the RyR-2 hyperactivity [57]. Dantrolene is a ryanodine receptor
antagonist that acts directly on the RyR-1 and RyR-3 to reduce channel
activation by CaM. Thereby it decreases the high Ca2+ concentration that leads
to sustained pathological muscle contraction[51, 53, 58]. Dantrolene also
stabilizes RyR-2 tetrameric structure and improves survival after myocardial
infarction[59].
Chemistry and Pharmacokinetics
Dantrolene is highly lipophilic and therefore poorly soluble in water.
Dantrolene can now be administered intravenously. One bottle of 20 mg
dantrolene sodium contains 3g mannitol to improve water solubility. The powder
should be dissolved in 60 ml water to achieve a concentration of 0.33mg/ml,
and adjusted the final pH to 9.5.
In 2014, , FDA approved Ryanodex, a new IV formulation of dantrolene
nanoparticles. Ryanodex (Eagle)[60] contains 250 mg of dantrolene as a
lyophilized powder. Each vial of powder requires only 5ml of sterile water (SWI)
to reconstitute for injection, while other previously approved formulations
required 60ml of SWI to reconstitute a 20 mg dose. Ryanodex contains less
mannitol, so patients may need to supplement with mannitol to produce more
urine[60].
Approximately 70% of dantrolene is absorbed after oral administration, with
a peak plasma time of six hours. However, plasma concentration varies
significantly from patient to patient, especially in children[61].The plasma
elimination half-life is estimated to be 12 hours. In children, the pharmacokinetic
profile is similar, with a half-life of approximately 10 hours [62]. Hepatic
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microsomes metabolize Dantrolene to 5-hydroxydantrolene. Dantrolene and its
metabolites are mainly excreted through urine and bile[63, 64].
Therapeutic Uses
In addition to malignant hyperthermia, dantrolene is also used to treat
other diseases in clinical work.
Muscle Spasm
The first approved use of oral dantrolene was for the treatment of
spasticity[65]. Spasticity is a common symptom of many neurological disorders,
such as cerebral infarction and MS[66]. Non-drug treatments for spasticity
include passive movements, exercises, posture and standing. Commonly used
anti-spasmodic drugs are those that act on the gamma aminobutyric acid
(GABA) energic system (baclofen, gabapentin, and benzodiazepines), the α-2
adrenergic system (tizanidine) Unlike other drugs that treat spasm, dantrolene
works directly on the muscle and so is less sedative.
Neuroleptic malignant syndrome
The incidence of neuroleptic malignant syndrome (NMS) has been reported
to be rare, and the most obvious clinical features of NMS are muscle rigidity
and hyperthermia. As the cause of altered thermoregulation remains unclear,
skeletal muscle contraction may generate heat. Therefore, paralytics will be a
key cooling measures[67]. Dantrolene, along with dopamine agonists such as
bromocriptine and amantadine, may provide benefits. However, these findings
are solely based on case reports due to the absence of controlled clinical
trials[68].
Ecstasy intoxication
One of the crucial signs of ecstasy intoxication is hyperthermia, which may
be caused by overstimulation of central serotonin. Dantrolene has been
proposed as a potentially effective treatment for ecstasy poisoning in
emergency rooms. However, recent animal studies suggest that dantrolene
sodium formulations are mechanistically unsuitable for the treatment of MDMA-
and METH-induced hyperthermia[69]
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Exertional heat stroke
Exertional heat stroke (EHS) is an acute injury with high morbidity and
mortality that is common in military and special operations environments. The
most effective way to treat EHS is immediately cooling. Dantrolene is
administered to the victim to prevent further muscle contractions and
hyperthermia. However, these data come from case reports. Whether
dantrolene treatment is beneficial for EHS still requires randomized, controlled
clinical studies[70].
Adverse Effects
Adverse effects may occur following acute or chronic parenteral
administration of dantrolene. The most common side effect was muscle
weakness (22%), followed by phlebitis (10%), respiratory failure (3%), and
gastrointestinal discomfort (3%). Other adverse symptoms of dantrolene
treatment include drowsiness, dizziness, and confusion[61]. Long-term oral
therapy at high dose has been associated with liver dysfunction, but dantrolene
is not the only potentially hepatotoxic substance administered to patients
reported to have this complication [65].
Dantrolene in Neurodegenerative Diseases
Dantrolene’s anti-inflammation effect
In recent years, research on application of dantrolene in different diseases
has been studied, including in vitro cell models and in vivo animal models.
Recent evidence suggests that disruption of intracellular Ca2+ homeostasis
resulting from overreaction of RyRs on the ER and/or SR, plays a crucial role
in sepsis and COVID-19-induced inflammation[71, 72]. Dantrolene, as a RyRs
antagonist, has been expected to improve SARS-CoV-2-mediated
inflammation and inhibit SIRS during sepsis by suppressing the concentration
of IL-6, IL-8, IL-1β, TNF-α, and IFN-γ3 in plasma and tissues[71, 72].
Wenk and his colleagues infused LPS, a commonly used inflammation
inducer, into the brain of 3-months-old F-344 AD rat for 28 days. At the
beginning of the inflammation induction, rats were treated subcutaneously with
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dantrolene (5 mg/kg/day) or nimodipine (5 mg/kg/day). They found that
treatment with nimodipine or dantrolene restored the number of TH-
immunoreactive cells in the locus coeruleus (LC) and significantly reduced
microglia activation in the hippocampus[73] and substantia nigra pars
compacta[74]. Furthermore, dantrolene treatment decreased inflammatory
gene expression of TLR4, iNOS, and TGFβ in the hippocampus, resulting in
neurotoxicity[73].
In another study of autoimmune disease, Fomina’s team used a mouse
model of experimental autoimmune encephalomyelitis (EAE), a T cell-mediated
autoimmune neuroinflammatory disease. They found that daily intraperitoneal
injection of dantrolene at 5 or 10 mg/kg beginning at the time of EAE induction
significantly reduced dampened inflammation in the spinal cord[75]. These
findings indicate that dantrolene has anti-inflammation effects in CNS.
Dantrolene’s therapeutic effects in AD and HD
In 2012, Professor Wei’s lab first reported the use of dantrolene for early
and long-term treatment in a murine AD model. Their team published a series
of articles on long-term treatment with dantrolene in both 3xTg and 5XFAD
mouse models[14, 15]. Their research also revealed that dantrolene reduced
amyloid accumulation in neurons by up to 76% compared to the vehicle control
and tended to reduce hippocampal phosphorylated tau protein[76]. Their
studies showed that dantrolene can ameliorate cognitive dysfunction in vivo and
has tolerable side effects with long-term use (treatment up to 10 months)[14].
In in vitro experiments on pluripotent stem cells, they found that dantrolene
could ameliorates the impairment of neurogenesis and synaptogenesis[16].
In recent years, impaired neural-lysosomal and autophagy-mediated
degradation of cellular debris has been suggested to contribute to neuritic
dystrophy and synaptic loss. Mustaly-Kalimi’s group demonstrated that
dantrolene can restore autophagic clearance of intracellular protein aggregates
by increasing vATPase levels, lysosomal acidification, and proteolytic activity
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in human iPSC-derived neurons from AD patients. Their work showed that
abnormal upstream Ca2+ dysregulation contributes to the pathological
accumulation of intracellular protein aggregates before the development of
overt histopathological or cognitive deficits in AD [77]. Studies also found that
dantrolene preserved synaptic plasticity. Zhang et al. used PS1-M146V knock-
in (KI) FAD mice with impaired long-term LTP maintenance (L-LTP). They found
that dantrolene could reverse insufficient mushroom spines maintenance in KI
neurons[78].
Besides these exciting findings, there are also controversial results. A study
genetically inhibiting RyRs expression in APPPS1 mice showed that blocking
RyR-3 was beneficial in older AD neurons while causing more synaptic
dysfunction in young AD neurons[56].
Altogether, these studies of dantrolene in AD are inspiring, but the
mechanisms by which dantrolene as a neuroprotective agent still require further
investigation.
HD is a progressive neurodegenerative disease caused by polyglutamine
amplification in the Huntington protein, resulting in selective degeneration of
spine neurons in the striatum. Bezprozvanny’s team fed yeast artificial
chromosome transgenic mice (YAC128), a model of HD, dantrolene (5 mg/kg)
twice a week from 2 months to 11.5 months of age. Their results showed that
the treatment group performed significantly better on beam-walking and gait-
walking tests. Furthermore, they performed neuropathological analysis on
YAC128 mice and found that the loss of NeuN-positive striatal neurons was
significantly reduced after long-term feeding of dantrolene[79].
Michael S and his colleagues combined whole-cell patch clamp and Two-
Photon Ca2+ Imaging to record action potential-evoked Ca2+ transients in
cortical pyramidal neurons (CPNs) in the R6/2 mouse model of juvenile HD at
different stages of disease progression. They demonstrated that in the
presence of dantrolene, the amplitude and area of Ca2+ transients were
significantly reduced compared with WT CPNs. The results suggest that
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dantrolene is a potential therapeutic agent for HD.
Potential mechanism of dantrolene ’s neuroprotective effects
Figure 1. Pyroptosis. Glutamate excitotoxicity result in pathological and
excessive activation of NMDAR and/or AMPAR, leading to pathological Ca2+
influx into cytosol space, increasing cytosolic and mitochondia Ca2+
concentration, mitochondrial reactive oxygen species (ROS) and then cytosol
ROS. Over activation of RyR results in excessive Ca2+ release from ER and
subsequent ER stress. The upstream Ca2+ dysregulation eventually results in
activation of NLRP3 inflammasome, activated caspase-1 and cleaved GSDMD
to generate N terminal GSDMD and releasing IL-1beta and IL-18 to generate
pathological inflammation.
RyRs are located on the sarco/endoplasmic reticulum (SR/ER) of nearly all
cell types. As we summarized previously, the mechanism of NDs is cell death
caused by mitochondrial dysfunction. Increasing studies show that aberrant
Ca2+ release from intracellular stores (SR/ER) is thought to be an upstream
cause of mitochondrial dysfunction. Therefore, aberrant Ca2+ release from
intracellular stores appears to be a key point.
Calcium is an extremely important signaling ion, transmitting cell signals for
various external stimuli. Under physiological conditions, more than 99% of
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intracellular Ca2+ is bound to cytosolic proteins or stored in the ER. Free
cytosolic Ca2+ is normally maintained at approximately 100 nm, but stimulation
can lead to an overall increase to approximately 1 μM[81]. The mechanisms
by which free cytosolic Ca2+ rises are Ca2+ entry through plasma membrane
channels and intracellular pools, in which ER plays an important role. As early
as 1961, Engstrom’s group first discovered that mitochondria could rapidly and
efficiently take up large amounts of Ca2+ when exposed to Ca2+ pulses, [82] . In
resting neurons, mitochondrial total Ca2+ and free Ca2+ levels are low, estimated
to be approximately 0.1 mM and 100 nM, respectively. However, upon
stimulation, mitochondria are able to accumulate enormous amounts of Ca2+[83,
84]. Elevated Ca2+ in mitochondria has many physiological effects, which are
especially important for cellular function. When Ca2+ concentration increases,
aerobic ATP production will be adjusted, and ROS production will be regulated
accordingly. Additionally, elevated Ca2+ contributes to synaptic transmission
and excitability, organelle dynamics, and activation of the release of death
signals[85, 86]. Accordingly, dantrolene (a RyRs inhibitor), has been shown to
block the release of ER Ca2+ stores and partially protect neurons from oxygen-
glucose deprivation toxicity [87]. Inhibiting the release of ER Ca2+ stores may
be one of the important mechanisms of dantrolene’s neuroprotective effects.
Chronic inflammation leads to excess Ca2+ to be transferred from the ERs
to mitochondria, causing mitochondrial calcium overload and further damaging
mitochondria[88]. Moreover, calpains are intracellular Ca2+- dependent
cysteine proteases that play a physiologic role by cleaving several substrates.
Overactivation of calpains can lead to changes in hippocampal structure and
function, and is linked to neuronal death. Furthermore, calpain is overactivated
by increased Ca2+ concentrations[89]. Hence, inhibiting the release of Ca2+
from the SRs may be one of the mechanisms by which dantrolene suppresses
the neuroinflammation.
Glutamate is the primary excitatory neurotransmitter in CNS, and one of the
glutamate receptors is NMDAR, which plays an important role in excitotoxic
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injury. Under physiological conditions, once NMDAR are activated, Na+ and
Ca2+ flow will be permitted, which is essential for normal synaptic transmission
as well as for a wide range of Ca2+-dependent signaling pathways. However,
significantly increased glutamate concentration, such as in stroke, leads to loss
of ion homeostasis and necrotic cell death by triggering massive NMDAR
stimulation [90]. The elevation of intracellular Ca2+ concentration triggers the
release of Ca2+ from ER through RyRs on ER[91]. Elevated intracellular Ca2+
levels lead to mitochondrial Ca2+ overload, which initiates a neurotoxic cascade
through multiple mechanisms, including mitochondrial membranes rupture,
ROS generation and release of cytochrome c and other proapoptotic factors[92].
Ultimately these mitochondrial-related events result in necrotic or apoptotic-like
cell death[93]. Furthermore, emerging studies show that abnormal Ca2+ release
from the ER/SR through RyRs Ca2+ channels play an important role in
neuroinflammation[94].
Taken together, dantrolene, a RyRs antagonist acts as a neuroprotective
agent through multiple mechanisms proposed below.
Ⅰ. Dantrolene protects neurons by decreasing excitotoxicity
In in vitro experiment on mouse cerebral cortex neurons, dantrolene reduced
glutamate-induced increases in intracellular Ca2+ by 70% under physiological
conditions, protecting neurons from glutamate-induced neurotoxicity[95]. In
another animal experiment, dantrolene significantly reduced the ischemia-
induced increase in glutamate concentration and prevented neuronal loss in the
CA1 region of the rat hippocampus [96]. In several other in vitro experiments,
dantrolene has been shown to inhibit activation of the NMDA receptors in
multiple different cell lines [97-99].
Ⅱ. Dantrolene protects neurons by decreasing apoptosis
Several animal models have showed neuroprotective effect through apoptosis
mechanism. In an in vivo experiment using repeated electroconvulsions as a
rat status epilepticus model, the TUNEL method was used to detect cell
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apoptosis. More TUNEL (+) neurons were observed in the hippocampus of the
dantrolene-treated group compared with the control group[100]. In another rat
spinal cord injury model, the author combined riluzole and dantrolene for
treatment; they found that rats treated with a combination of riluzole and
dantrolene showed a greater number of NeuN-positive neurons, suggesting
that dantrolene prevents neuronal apoptosis [101].
An in vitro experiment using neuron-like PC12 cells demonstrated that
dantrolene, as well as 2-aminoethoxydiphenyl borate (2-APB), another RyRs
and InsP3 receptor (InsP3R) inhibitor, protect PC12 cells from H2O2-induced
apoptosis and activate autophagic pathways[102].
Ⅲ. Dantrolene protects neurons by decreasing neuroinflammation
An increasing number of studies, including different animal models of NDs
show that Pro-inflammatory markers are significantly reduced after treatment
with dantrolene as we mentioned before. The proposed mechanisms by which
dantrolene decreases neuroinflammation include the following approaches.
1. Dantrolene reduces neuroinflammation by inhibiting microglia activation
Neuroinflammation is characterized by the production of pro-inflammatory
cytokines, including IL-1β, IL-6, IL-18 and tumor necrosis factor (TNF), in which
microglia play an important role. Once the microglia are activated by
pathological factors such as apoptotic neurons or infectious pathogens, they
produce increased amounts of pro-inflammatory cytokines[103]. Furthermore,
the insufficient phagocytic capacity of activated microglia leads to the inefficient
clearance of protein aggregates such as Aβ in AD[22]. Activated microglia can
also directly eliminate synaptic structures[103]. The emerging role of microglial
activation in the pathogenesis of NDs makes microglia a therapeutic target.
Ca2+ is required for LPS-mediated microglia activation in vitro, and the
application of Ca2+ chelators is sufficient to prevent activation and production of
proinflammatory cytokines[104]. A study has shown that nitrogen-doped
graphene quantum dots (N-GQDs) can induce intracellular calcium overload by
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activating calcium channels of L VGCCs and RyRs, thereby activating mouse
hippocampal microglia. [105]. These evidences revealed that RyRs and L-
VDCCs participated in mediating Ca2+-related microglia activation. Microglia
express mRNA for RyR1 and RyR2 isoforms, and application of RyRs
antagonists prevents microglia-mediated LPS-induced neurotoxicity [106].
Another study demonstrated that dantrolene significantly reduced the number
of activated microglia in the hippocampus and reduced the expression of
various pro-inflammatory cytokines[73]. Taken together, dantrolene decrease
microglia activation by reducing intracellular calcium overload through RyRs
channels.
2. Dantrolene reduces neuroinflammation by inhibiting pro-inflammatory
cytokines
As we summarized previously, most NDs are characterized by a
pathological inflammatory response. The inflammatory response is initially
protective, but subsequently, inflammatory cytokines such as TNFα, IL-6,
interleukin 1α(IL-1α), and GM-CSF[22] lead to cell death and tissue damage
[34]. Intracellular Ca2+ signaling and the elevation of intracellular Ca2+ are
critical for the release of pro-inflammatory cytokines [71]. Furthermore, calcium
channels contribute to the release of pro-inflammatory cytokines in NDs. A
recent study showed that knockdown of transient receptor potential vanilloid
4(TRPV4), a nonselective Ca2+ channel, can reduce the concentration of pro-
inflammatory cytokines including IL-18, COX-2, and 5-LOX in PD mice[107].
The calcium channel blocker carvedilol has been demonstrated to decrease the
release of pro-inflammatory cytokine TNF-α IL-2 and IL-6[108]. Many research
groups also reported that VGCCs and RyRs play an important role in
neuroinflammation[68, 109].
Dantrolene, a calcium channel blocker with the ability to ameliorate Ca2+
dysregulation by inhibiting RyRs, has been discovered to suppress plasma and
tissue concentration of IL-6, IL-8, IL-1β, TNF-α, and IFN-γ36 in vivo and in
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vitro[110-113]. Taken together, dantrolene inhibited ER-mediated Ca2+ release
and reduced pro-inflammatory cytokine release.
3. Dantrolene reduces neuroinflammation by ameliorating oxidative stress
Oxidative stress (OS) and neuroinflammation are two different but
fundamental pathological factors that play important roles in the onset and
progression of NDs [114]. Inflammatory cells such as microglia and astrocytes
secrete reactive substances that promote OS [114]. Some ROS and RNS
(reactive nitrogen species) can reversely stimulate intracellular signaling
cascades, resulting in an increased release of pro-inflammatory cytokines.
Therefore, neuroinflammation and OS can promote each other, especially in
the NDs state.
Given that oxidative stress plays a crucial role in NDs by causing cell death
and promoting neuroinflammation, antioxidant drugs have been a potential
treatment for NDs. Dantrolene has been reported to protect cells from oxidative
stress by increasing the concentration of GSH and GSH/GSSG [115, 116].
Furthermore, calcium influx through RyRs on ER is associated with ROS
generation [117].
4. Dantrolene reduces neuroinflammation by ameliorating pyroptosis
Pyroptosis is a type of programmed cell death during which the
inflammatory cytokines are released. Pyroptosis is mediated by GSDMD-N (N-
terminal fragment of gasdermin D) which binds to membranes to form
membrane pores and promotes the release of inflammatory cytokines,
especially IL-1β. NLRP3 inflammasome activation plays a leading role in the
initiation of this process. Pyroptosis is closely related to neuroinflammatory
diseases, including subarachnoid hemorrhage, spinal cord injury [118] and PD
[119]. Recent studies have shown that ER stress and excessive Ca2+ release
are associated with NLRP3 inflammasome activation. A recent study shows
that restriction of Ca2+ release in the ER decreases the NLRP3 inflammasome
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activation[118]. Dantrolene, as a RyRs antagonist, can also inhibit Ca2+ release
in the ER and therefore, potentially ameliorate pyroptosis by inhibiting NLRP3
inflammasome activation.
Future trends in NDs and potential application of dantrolene
In summary, the pathogenesis of NDs primarily involves
neuroinflammation and apoptosis mediated by mitochondrial dysfunction. To
date there are no disease-modified drugs. On the one hand, dantrolene shows
anti-inflammatory effect in a variety of NDs; on the other hand, there are
disease-modified drugs for the treatment of AD. All these evidences suggest
that dantrolene, as a potential anti-inflammation drug, can be considered as a
potential therapeutic drug for the treatment of different NDs.
CRediT authorship statement
Conceptualization: Wenila Zhang, Huafeng Wei; Data curation: Xu Zhao ;
Formal analysis: Xu Zhao ; Investigation: Piplu Bhuiyan ; Methodology: Piplu
Bhuiyan; Project administration: Huafeng Wei; Supervision: Huafeng Wei;
Validation: Huafeng Wei; Writing – original draft: Wenila Zhang; Writing –
review & editing: Henry Liu. All authors have read and agreed to the published
version of the manuscript.
Disclosure statement
Not applicable.
Ethical statement
Not applicable.
Data availability statement
All study data are included in the article.
Funding
This work was supported by grants to HW from the National Institution of
Aging (R01AG061447, 3R01AG061447- 03S1).
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Acknowledgments
The author would like to thank Luna Sato and Yutong Yi for English editing
the manuscript.
Declaration of competing interests
Henry Liu holds the position of Editor-in-Chief for JATM, and was blinded from
reviewing or making decisions for the manuscript. Dr. Huafeng Wei is one of
the inventors for patents application titled "Intranasal dantrolene for treatment
of Alzheimer's disease" owned by the University of Pennsylvania Trustee in
following countries: International (PCTUS2020/040198, 2020), USA
(62/868,820, 2019), Europe (20833145.4-1112, 2022), Japan (2021-577376,
2021), China (202080054348.2, 2022), Hong Kong (62022053033.60, 2020),
South Korea (10-2022-7003375, 2020), Singapore (11202114347V, 2020),
Brazil (BR1120210265970, 2020), Mexico (MX/a/2022/000231, 2022). The
authors declare that they have no conflicts of interest.
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