Modulating ryanodine receptors with dantrolene attenuates neuronopathic phenotype in Gaucher disease mice

Abstract

Neuronopathic Gaucher disease (nGD) manifests as severe neurological symptoms in patients with no effective treatment available. Ryanodine receptors (Ryrs) are a family of calcium release channels on intracellular stores. The goal of this study is to determine if Ryrs are potential targets for nGD treatment. A nGD cell model (CBE-N2a) was created by inhibiting acid β-glucosidase (GCase) in N2a cells with conduritol B epoxide (CBE). Enhanced cytosolic calcium in CBE-N2a cells was blocked by either ryanodine or dantrolene, antagonists of Ryrs and by Genz-161, a glucosylceramide synthase inhibitor, suggesting substrate-mediated ER-calcium efflux occurs through ryanodine receptors. In the brain of a nGD (4L;C*) mouse model, expression of Ryrs was normal at 13 days of age, but significantly decreased below the wild type level in end-stage 4L;C* brains at 40 days. Treatment with dantrolene in 4L;C* mice starting at postnatal day 5 delayed neurological pathology and prolonged survival. Compared to untreated 4L;C* mice, dantrolene treatment significantly improved gait, reduced LC3-II levels, improved mitochondrial ATP production and reduced inflammation in the brain. Dantrolene treatment partially normalized Ryr expression and its potential regulators, CAMK IV and calmodulin. Furthermore, dantrolene treatment increased residual mutant GCase activity in 4L;C* brains. These data demonstrate that modulating Ryrs has neuroprotective effects in nGD through mechanisms that protect the mitochondria, autophagy, Ryr expression and enhance GCase activity. This study suggests that calcium signalling stabilization, e.g. with dantrolene, could be a potential disease modifying therapy for nGD.

Basal ryanodine receptor activity suppresses autophagic flux

“…These findings were supported by experiments performed in differentiated C2C12 cells and in primary dissociated hippocampal neurons, two cell types that express endogenous RyRs. (27) Hence, these results indicate that RyR-mediated Ca 2+ -release events suppresses autophagic flux at steps distal to the initiation of phagophore formation, and involving alteration in lysosomal function…”

“…(26) Second, it was reported that in a mouse model of neuropathic Gaucher disease RyR inhibition, using dantrolene, delayed the neurological pathology and increased survival. (27) The authors reported that dantrolene could partially reverse the observed alterations in autophagic flux, suggesting a possible role for RyRs in regulating autophagy. However, it was not further studied how RyRs influenced autophagy.…”

Ion channels in the regulation of autophagy

“…73 Further, the RYR antagonist dantrolene reduces LC3-II levels in the brain of neuronopathic Gaucher disease mice exhibiting abnormal autophagy. 74 In accordance with this, a very recent study showed that basal RYR3 activity suppresses autophagic flux at the level of lysosomes. Specifically, pharmacological inhibition of endogenous (in C2C12 myoblasts and primary hippocampal neurons) or ectopically expressed (in HEK293 cells) RYRs augments autophagic flux by increasing lysosomal turnover.…”

Pre-clinical Mouse Models of Neurodegenerative Lysosomal Storage Diseases

“…Pre-clinical trials evaluating receptor antagonism (RA), AMPA-RA (Kovacs and Pearce, 2008) and NMDA-RA (Finn et al, 2013) have improved the neurobehavioral phenotypes associated with LSDs. Furthermore, a number of pharmacological agents have been explored eliciting various effects from modulating membrane fluidity (Schultz et al, 2018), modulating Ca 2+ (Chang et al, 2007) or cholesterol levels (Erickson et al, 2000;Pelled et al, 2003;Kim et al, 2007;Repa et al, 2007;Abi-Mosleh et al, 2009;Liu et al, 2010;Taylor et al, 2012;Hovakimyan et al, 2013;Nusca et al, 2014;Tanaka et al, 2014;Soga et al, 2015;Demais et al, 2016;Liou et al, 2016), and enhancing enzyme activity (Arroyo et al, 2014) via the use of neurosteroids (NS) (Griffin et al, 2004;Liao et al, 2009). There is also interest in facilitating sphingolipid degradation via the upregulation of heat shock proteins (HSP) (Chung et al, 2016;Kirkegaard et al, 2016).…”

“…(pmuts) (Xu et al, 2003) + + Gba (lnl/lnl)(Enquist et al, 2007) + + Chaperone(Dasgupta et al, 2015), CM(Liou et al, 2016), ERT (Cabrera-Salazar et al, 2010), SRT (Cabrera-Salazar et al, 2012)GLB1GM1-GangliosidosisGlb1 (−/−)(Matsuda et al, 1997) + + Chaperone(Takamura et al, 2011) …”