Elsevier

Neuropharmacology

Volume 110, Part A, November 2016, Pages 211-222
Neuropharmacology

1-Deoxysphingolipid-induced neurotoxicity involves N-methyl-d-aspartate receptor signaling

https://doi.org/10.1016/j.neuropharm.2016.03.033Get rights and content

Highlights

  • 1-deoxysphinganine (1-deoxySA) triggers neuronal cytoskeletal alterations and deregulation of associated proteins.

  • 1-deoxySA activates the N-methyl-D-aspartate receptor (NMDAR) but down regulates GluN2B and PSD-95 expression.

  • 1-deoxySA-induced neurotoxicity can be prevented via NMDAR inhibition or blocking ceramide synthesis.

  • Cytoskeletal stabilization and/or glutamate receptor inhibition may potentially prevent 1-deoxySA-induced neurodegeneration.

Abstract

1-Deoxysphingolipids (1-deoxySL) are atypical and neurotoxic sphingolipids formed by alternate substrate usage of the enzyme serine-palmitoyltransferase. Pathologically increased 1-deoxySL formation causes hereditary sensory and autosomal neuropathy type 1 (HSAN1) - a progressive peripheral axonopathy. However, the underlying molecular mechanisms by which 1-deoxySL acts are unknown. Herein we studied the effect of 1-deoxysphinganine (1-deoxySA) and its canonical counterpart sphinganine (SA) in aged cultured neurons comparing their outcome on cell survival and cytoskeleton integrity. 1-deoxySA caused rapid neuronal cytoskeleton disruption and modulated important cytoskeletal regulatory and associated components including Rac1, Ezrin and insulin receptor substrate 53. We show that 1-deoxySA is internalized and metabolized downstream to 1-deoxydihydroceramide since inhibition of ceramide synthase protected neurons from 1-deoxySA-mediated cell death. In addition, 1-deoxySA reduced protein levels of N-methyl-d-aspartate receptor (NMDAR) subunit GluN2B, the postsynaptic density protein 95 and induced cleavage of p35 to p25. Notably, blocking NMDAR activation by MK-801 or memantine significantly prevented 1-deoxySA neurotoxicity. Functional studies of differentiating primary neurons via the patch-clamp technique demonstrated that 1-deoxySA irreversibly depolarizes the neuronal membrane potential in an age-dependent manner. Notably, only neuronal cells that displayed functional NMDAR- and NMDA-induced whole-cell currents responded to 1-deoxySA treatment. Furthermore, pre-exposure to the non-competitive antagonist MK-801 blocked the current response of NMDA and glycine, as well as 1-deoxySA. We conclude that 1-deoxySA-induced neurotoxicity compromises cytoskeletal stability and targets NMDAR signaling in an age-dependent manner. Thus stabilization of cytoskeletal structures and/or inhibition of glutamate receptors could be a potential therapeutic approach to prevent 1-deoxySA-induced neurodegeneration.

Introduction

Sphingolipids participate in plasma membrane formation but are also important signaling molecules in several cellular processes (Ghosh et al., 1990, Obeid et al., 1993, Sim-Selley et al., 2009). The first step of de novo sphingolipid biogenesis is catalyzed by the serine-palmitoyltransferase (SPT) that conjugates palmitoyl-CoA with l-serine forming the long chain base sphinganine (SA). To a certain extent SPT can also use alanine or glycine as alternative substrates forming a class of atypical 1-deoxy-sphingolipids (1-deoxySL). Conjugation with alanine forms 1-deoxysphinganine (1-deoxySA) whereas the use of glycine forms 1-deoxymethylsphinganine. Both metabolites lack the C1-hydroxyl group of the canonical substrate sphinganine (Zitomer et al., 2009). 1-DeoxySA is converted to 1-deoxy(dihydro)ceramides and 1-deoxysphingosine but not metabolized to complex sphingolipids, nor canonically degraded as the catabolic intermediate sphingosine-1-phosphate cannot be formed (Bertea et al., 2010). 1-DeoxySL induce cell death in various cells types (Cuadros et al., 2000, Salcedo et al., 2007, Sanchez et al., 2008, Zuellig et al., 2014) and are neurotoxic for isolated dorsal root ganglion neurons inducing neurite retraction and alterations in neurofilament structure (Penno et al., 2010, Jun et al., 2015).

Hereditary sensory and autosomal neuropathy type 1 (HSAN1) is a dominantly inherited axonal neuropathy associated with several missense mutations in the SPT genes SPTLC1 and SPTLC2. These mutations greatly increase the irregular activity of SPT with alanine and glycine resulting in significantly enhanced deoxySL formation (Penno et al., 2010, Eichler et al., 2009, Gable et al., 2010). Clinically, HSAN1 is characterized by a progressive loss of pain and temperature sensation, neuropathic pain attacks and skin ulcers (Houlden et al., 2006), characteristics that resemble the features of diabetic sensory neuropathy (DSN). Increased 1-deoxySL formation was also reported for patients with metabolic syndrome and diabetes mellitus type 2 (T2DM) (Bertea et al., 2010, Othman et al., 2012) suggesting that deoxySLs may also be involved in DSN pathology. Since the molecular pathways that drive 1-deoxySA-induced neurotoxicity are largely unknown we investigated its mechanism of action on survival and cytoskeletal integrity of cultured primary neurons. Our data show that altered NMDAR activity may contribute to 1-deoxySA-mediated neuropathology.

Section snippets

Material and methods

All experiments were performed in accordance with Swiss animal protection laws and University of Zürich institutional guidelines for animal experimentation.

1-DeoxySA is neurotoxic

First, we analyzed the effect of 1-deoxysphinganine (1-deoxySA) on neuronal survival. After culture for 17 days in vitro (DIV) neurons were treated with either 1-deoxySA, C18SA or C18SO for 24 h or 48 h and cell death was monitored using MTT and LDH cytotoxicity assays (Fig. 1). Exposure to 1-deoxySA (0.5 μM) for 24 h decreased mitochondrial activity and a further reduction was observed after 48 h (52.7% ± 24.0, p < 0.001 and 15.0% ± 5.7, p < 0.001 respectively; Fig. 1A). At 2 μM, 1-deoxySA

Discussion

Neurotoxic 1-deoxyspingolipids are involved in the pathology of HSAN1 and may also contribute to the progression of T2DM and DSN (Bertea et al., 2010, Zuellig et al., 2014, Othman et al., 2012). This work shows 1-deoxySL-induced neuronal death is likely mediated by 1-deoxy-ceramide species and identifies several cytoskeleton-associated factors modulated by 1-deoxySA. Moreover, 1-deoxySA activates the NMDAR and NMDAR inhibition greatly prevents 1-deoxySA-induced neuronal death. Functional

Funding

This work was financed by grants from Zurich Center of Integrated Human Physiology, University of Zurich (ZIHP); the 7th Framework Program of the European Commission (“RESOLVE”, Project number 305707) and Rare Disease Initiative Zurich (“RADIZ”), University of Zurich.

Conflicts of interest

None.

Acknowledgements

We thank Shalmali Patkar for performing NMDAR RT-PCR, Anna Bogdanova for the NMDAR antagonists and Thomas Lutz and Heiko Bode for valuable discussions.

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