Dantrolene, A Treatment for Alzheimer's Disease?

Abstract

Alzheimer's disease (AD) is a fatal progressive disease and the most common form of dementia without effective treatments. Previous studies support that the disruption of endoplasmic reticulum (ER) Ca²⁺ via overactivation of Ryanodine receptors (RYRs) plays an important role in the pathogenesis of AD. Normalization of intracellular Ca²⁺ homeostasis could be an effective strategy for AD therapies. Dantrolene, an antagonist of RYRs and an FDA approved drug for clinical treatment of malignant hyperthermia and muscle spasms, exhibits neuroprotective effects in multiple models of neurodegenerative disorders. Recent preclinical studies consistently support the therapeutic effects of dantrolene in various types of AD animal models and were summarized in the current review.

Alzheimer's disease (AD), a fatal progressive disease and the most common form of dementia, threatens around 24 to 35 million people worldwide.^1-5 It is estimated that this disease affects around 6 % of population aged over 65 years, with its incidence increasing with age. Patients suffer memory loss and cognitive function decline, and on average die nine years after diagnosis.^6,7

Amyloid cascade hypothesis of AD is a prominent idea in the research field of AD pathogenesis.^8-12 It arises from the observation that patients affected by AD are characterized by the accumulation of senile plaques containing a product of amyloid precursor protein (APP) metabolism called the Amyloid beta (Aβ) peptide.^13-15 Histopathological and genetic evidence form the basis of the amyloid cascade hypothesis, which states that deposition of Aβ is the initiating event that triggers neuronal dysfunction and death.^16-18 Aβ peptides constitute a major part of the neuritic plaques causing neurotoxicity, which are cleaved from APP by β- and γ-secretases.¹⁹ Hitherto, much of the research focus in the AD field has been on Aβ peptide generation and its mechanisms of action.^20-22 Despite the efforts to characterize the molecular mechanisms underlying Aβ's toxicity, it remains unclear what triggers the accumulation of the peptide and whether such action is the primary cause of AD pathogenesis and cognitive dysfunction.^18,23-26 However, the amyloid cascade hypothesis still dominates the search for AD disease treatments up to now. For example, researchers have tried to reduce Aβ production by developing molecules that inhibit γ-secrease activity, block Aβ aggregation and promote Aβ clearance.^27-32 Unfortunately, none of the amyloid-targeting molecules that reached clinical trials have succeeded, despite of decades of basic and clinical research.

Calcium is one of the most important second messengers³³ in the nervous system because it plays an essential role in wide range of cellular function including learning and memory, synaptic activity and neurotransmitter action, excitotoxicity, and cell death.^34-37 Neurons maintain intracellular Ca²⁺ content mainly through Ca²⁺ signal transduction pathways. Neuronal Ca²⁺ influx is regulated by different Ca²⁺ channels, including voltage-dependent Ca²⁺ channels (VDCC), α-mino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptors, nicotinic receptors, N-methyl-D-aspartate (NMDA) receptors and store-operated Ca²⁺ channels (SOC).^38-40 Ca²⁺ can also be released from the primary intracellular stores of endoplasmic reticulum (ER).^41-45 Neurons are highly sensitive to any changes in intracellular Ca²⁺ concentrations: insufficient intracellular Ca²⁺ content leads to abnormal functioning of neurons, whereas excessive Ca²⁺ levels cause cell death.^40,46,47 Therefore, even small fluctuations of Ca²⁺ content can significantly change the physiological functions of cells.^39,48-51

In 1987, Dr. Zaven Khachaturian suggested the Ca²⁺ hypothesis of AD that disruption in intracellular Ca²⁺ homeostasis leads to the final common pathway for AD and age-associated brain changes.^48,52-55 This hypothesis is supported by the presence of AD-like symptoms in mouse models harboring presenilin's mutations and synaptic dysfunction due to ER Ca²⁺ concentration changes in the absence of Aβ pathology.^56-58 The basis of the Ca²⁺ hypothesis in AD is that the disruption of intracellular Ca²⁺ signaling homoeostasis contributed to both the progressive decline in memory and the increase in neuronal cell apoptosis.^59,60 Any neuropathology of AD, particularly sporadic AD, has to account for the slow progression of the disease and for the fact that the changes in synaptic physiology and onset of memory loss often precedes any evidence for the massive cell loss that characterizes the later stages of AD. Also, neuronal cell death were observed when Ca²⁺ levels exceeded the normal physiologic range, and the Ca²⁺-mediated signaling system is altered in the aging nervous system resulting in altered neuronal functioning and/or cell death.^61,62 In addition, Ca²⁺ dysregulation is further implicated in AD since each of the genes currently known to influence AD susceptibility so far (APP, PSEN1, PSEN2, and APOE) affects intracellular Ca²⁺ levels and/or calcium signaling.^63-65 Early changes in intraneuronal Ca²⁺ regulation may be also common observations in AD patients. Since intracellular Ca²⁺ homeostasis plays such an important role in both neuronal and synapse function, its disruption produce symptoms of brain aging, neuronal degeneration and death, synapse and cognitive dysfunction.^66-69 Up to date, the proposed mechanisms responsible for Ca²⁺ dysregulation in AD primarily include overactivation of ryanodine receptors and InsP3R, which may contribute to early AD neuropathology and susceptibility.^35,70-74

Two well studied Ca²⁺ channels release Ca²⁺ from the ER into cytosolic space: Inositol-1,4,5-trisphosphate receptors (InsP₃Rs) and Ryanodine receptors (RYRs).^75-78 As shown in figure 1, mutations in presenilin-1 (PS1) and presenilin-2 (PS2) associated with familial AD (FAD), significantly enhance the expression and activation of RYRs, as well as the activation of InsP₃Rs, resulting in excessive Ca²⁺ release from the ER and abnormal cytosolic Ca²⁺ concentration [Ca²⁺]_c elevation.^50,79-81 On the other hand, the ApoE4 gene, a widely accepted genetic risk factor for sporadic AD, has been shown to disrupt intracellular Ca²⁺ homeostasis by abnormally increasing NMDA receptor activation, which may result in excessive Ca²⁺ influx, elevation of [Ca²⁺]_c, and Ca²⁺-induced Ca²⁺ release (CICR) from the ER via RYRs and/or IP₃Rs.

Figure 1. Calcium dysregulation in AD and the proposed effects of dantrolene.

Mutated Presenilins (PS) in familial AD (FAD) increase numbers and activation of RYRs and activation of InsP₃Rs, resulting in excessive Ca²⁺ release from the ER. Overactivation of RYRs and subsequent disruption of intracellular Ca²⁺ homeostasis in AD can then cause the following pathological changes: 1). Increase presynaptic glutamate release and postsynaptic NMDA receptor activation and glutamate excitotoxicity; 2). Increase postsynaptic cytosolic [Ca²⁺]_c and neurodegeneration; 3). Form a vicious cycle between amyloid pathology and Ca²⁺ dysregulation; 4). Promote Tau pathology and damage of microtubules; 5). Synapse dysfunction. These above pathological changes may result in synaptic dysfunction, neurodegeneration and cognitive dysfunction. Dantrolene is expected to restore intracellular Ca²⁺ homeostasis, resulting in inhibition of neuropathology and cognitive dysfunction in AD.

RYRs are expressed in soma, proximal dendrites as well as in distal processes and spines. The three isoforms of RYRs are RYR1, RYR2 and RYR3, all of which are expressed in the central nervous system.^82-85 RYR1 is expressed in cerebellar Purkinje cells.^84,86,87 RYR2 is found in the olfactory nerve layer, dentate gyrus, cerebral cortex, cerebellar granule cells, the facial nucleus and the motor trigeminal nucleus.^39,88 Lastly, RYR3 is highly expressed in the hippocampal CA1 pyramidal cell layer, dorsal thalamus and caudate putamen.^49,85 Overactivation of RYRs in the brain and excessive Ca²⁺ release from the ER may result in increased excitatory glutamate release from presynaptic spaces, neuronal death, neurodegeneration, and Aβ pathologies, which is considered as early pathogenic factors in AD (Figure 1).^34,89-91 Thus, reducing Ca²⁺ over-release through the inhibition of RYRs is a reasonable way to mitigate the AD pathology and ameliorate the memory and cognitive problems.^74,92,93

Dantrolene is an FDA approved drug for clinical treatment of malignant hyperthermia, and muscle spasms, which is an antagonist of RYRs.^94-99 The most common side effects of dantrolene are dizziness, drowsiness, light headedness, headaches, anorexia, diarrhea, nausea, and vomiting. Chronic oral use can be associated with liver dysfunction. Rarely observed side effects are fatigue, weakness and rash.^39,77, Although the common side effects of dantrolene originate in the central nervous system, several studies addressed the beneficial effects of dantrolene in AD pathology via acting on RYRs in recent years.^{39,77,100-103} In April of 2012, Peng et al.¹⁰⁴ published a pioneer paper investigating the therapeutic effect of dantrolene on ethology and pathology in the triple transgenic Alzheimer mouse model (3xTg-AD). Wild type or 3xTg-AD mice from 2 to 13 months of age were treated with dantrolene continuously. Compared to control, 3xTg-AD mice treated with dantrolene exhibited significant improvement in memory retention and working memory. In fact, dantrolene treated 3xTg-AD mice performed the same memory and learning ability as the wild type control mice. In addition, there was no significant difference in motor function among all groups. Immunohistochemical analysis of phosphorylated GSK-3β and phosphorylated Tau in the cortex, and synaptic marker expression did not show any statistical significance among all treated groups. Notably, amyloid plaques in the hippocampus in dantrolene treated 3xTg-AD mice significantly reduced compared to its corresponding control group. Taken together, the results showed that early and chronic dantrolene treatment starting before the initiation of amyloid pathology significantly decreased amyloid plaque load in the hippocampus and memory deficits in 3xTg-AD mice.

In August of 2012, Oules et al.¹⁰⁵ showed that APP contributes to ER Ca²⁺ homeostasis and in turn ER Ca²⁺ could influence Aβ production. The authors also found that over expression of wild-type human APP (APP₆₉₅) in human SH-SY5Y neuroblastoma cells or the Swedish double mutation APP (APP_swe) in APP_swe-expressing mice (Tg2576) enhances RYR expression and increases ER Ca²⁺ release via RYRs. The authors' use of dantrolene to block RYRs decreased the release of RYR-mediated Ca²⁺ and contributed to the reduction of both intracellular and extracellular Aβ load in mouse Tg2576 primary cultured neurons and in human SH-SY5Y neuroblastoma cells. Also, Aβ in the hippocampus and cortex decreased in dantrolene-treated AD mice. The authors speculated that dantrolene reduces APP phosphorylation on Thr-668 residue through the regulation of RYR-mediated Ca²⁺ release by means of the modulation of GSk3β and Cdk5 kinase activities. Their study also notes that dantrolene decreases β- and γ-secretases activities. As a result, dantrolene prevents learning and memory decline by reducing C99 and Aβ production.

In December of 2012, Chakroborty et al.¹⁰⁶ reported sub-chronically short-term (4 weeks) treatment of AD models with dantrolene. Using patch clamp recordings and 2-photon Ca²⁺ imaging of hippocampal slices, the authors found that ER Ca²⁺ signaling is fully normalized in dendritic compartments in dantrolene treated early and later-stage AD mice. In addition, the increased RYR2 levels and enhanced IP₃R-mediated Ca²⁺ release in AD mice were restored to normal levels with dantrolene treatment. Thus, sub-chronic dantrolene treatment stabilizes RYRs' function and expression and inhibits abnormal CICR initiated through IP₃R-mediated Ca²⁺ release. These aforementioned studies suggest that inhibition of RYRs with dantrolene exerts beneficial effects on AD pathology.

However, not all laboratory findings on dantrolene treatment in AD are consistent. When Zhang et al.¹⁰⁷ investigated the role of presenilins in neuronal ER Ca²⁺ release in 2010, they found that the long-term oral dantrolene treatment of 2-8 month old AD (APP-PS1 mutant) mice increased amyloid load and neuronal atrophy in hippocampal and cortical regions along with loss of synaptic markers, raising some doubts about the therapeutic potential of dantrolene. It is not clear what the reasons are for the discrepancies among all these studies, but differences in mouse models, treatment duration, and route of administration may be contributing factors. Further studies in different AD animal models are urgently needed to investigate and confirm if dantrolene or other raynodine inhibitors will ameliorate or aggravate the neuropathology in AD.

In summary, excessive Ca²⁺ release from the ER modulates the amyloid genic processing pathway and other AD pathology, thereby promoting memory loss.^{43,57,74,108,109} Dantrolene, as an antagonist of RYRs on ER, mitigates AD pathology and may serve as a probe compound for future studies to treat Alzheimer's disease.

Abbreviations

AD

Alzheimer's disease

APP

amyloid precursor protein

Aβ

Amyloid beta

Ca²⁺

Calcium

VDCC

voltage-dependent Ca²⁺ channels

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid

NMDA

N-methyl-D-aspartate

SOC

store-operated Ca²⁺ channels

ER

endoplasmic reticulum

InsP₃Rs

Inositol-1,4,5-trisphosphate receptors

RYRs

Ryanodine receptors

CICR

cytosolic Ca²⁺ concentration [Ca²⁺]_c, Ca²⁺-induced Ca²⁺ release