Development of a model system for neuronal dysfunction in Fabry disease

Abstract

Fabry disease is a glycosphingolipid storage disorder that is caused by a genetic deficiency of the enzyme alpha-galactosidase A (AGA, EC 3.2.1.22). It is a multisystem disease that affects the vascular, cardiac, renal, and nervous systems. One of the hallmarks of this disorder is neuropathic pain and sympathetic and parasympathetic nervous dysfunction. The exact mechanism by which changes in AGA activity result in change in neuronal function is not clear, partly due to of a lack of relevant model systems. In this study, we report the development of an in vitro model system to study neuronal dysfunction in Fabry disease by using short-hairpin RNA to create a stable knock-down of AGA in the human cholinergic neuronal cell line, LA-N-2. We show that gene-silenced cells show specifically reduced AGA activity and store globotriaosylceramide. In gene-silenced cells, release of the neurotransmitter acetylcholine is significantly reduced, demonstrating that this model may be used to study specific neuronal functions such as neurotransmitter release in Fabry disease.

Fabry disease pain: patient and preclinical parallels

“…Models of Fabry disease have been developed in human embryonic kidney cells HEK and human neuroblastoma cells differentiated into cholinergic-like neurons. 81 , 99 In both cell types, α-Gal A RNA expression was knocked down using short hairpin RNAs. Both studies observed accumulation of Gb3 deposits.…”

“…Both studies observed accumulation of Gb3 deposits. 81 , 99 A-Gal A–deficient HEK cells exhibited an altered sodium channel function, and the neuroblastoma cells showed differences in proliferation and release of the neurotransmitter acetylcholine. 81 These cells are relatively easy to culture and manipulate; however, both HEK and neuroblastoma cells are phenotypically different from native sensory neurons and therefore are likely limited in modeling sensitization mechanisms in sensory neurons.…”

“… 92 However, despite the use of the Fabry mouse model to validate retention of the enzyme and monitor its effectiveness clearing Gb3, researchers have not extensively assessed its impact on any pain or sensory abnormalities. Although in vitro models can provide information on effectiveness of Gb3 clearance and potential effects of ion channels, 81 , 99 these cellular models cannot predict the actual effect on pain or pain behavior. Therefore, more preclinical animal work that examines the effects of ERT and other therapies on pain in vivo is needed to better understand why ERT in patients is not as effective in alleviating pain as expected.…”

Bridging the Gap Between Vessels and Nerves in Fabry Disease

“…Interestingly, studies also pointed to an altered sympathetic innervation as well as noradrenergic/cholinergic neurotransmitter coding in Fabry disease (Stemper and Hilz, 2003;Jardim et al, 2006;Kaneski et al, 2016). In this context, an in vitro model for neuronal dysfunction in Fabry disease suggested insufficiency of cholinergic function (Kaneski et al, 2016).…”

“…Interestingly, studies also pointed to an altered sympathetic innervation as well as noradrenergic/cholinergic neurotransmitter coding in Fabry disease (Stemper and Hilz, 2003;Jardim et al, 2006;Kaneski et al, 2016). In this context, an in vitro model for neuronal dysfunction in Fabry disease suggested insufficiency of cholinergic function (Kaneski et al, 2016). It was also shown that during local and whole-body heating, acetylcholine is released, inducing cutaneous vasodilation, presumably via NO synthase pathways (Shibasaki et al, 2002;Holowatz et al, 2005).…”