Neuronal overexpression of IP3 receptor 2 is detrimental in mutant SOD1 mice

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Abstract

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing progressive paralysis of the patient followed by death on average 3–5 years after diagnosis. Disease pathology is multi-factorial including the process of excitotoxicity that induces cell death by cytosolic Ca2+ overload. In this study, we increased the neuronal expression of an endoplasmic reticulum (ER) Ca2+ release channel, inositol 1,4,5-trisphosphate receptor 2 (IP3R2), to assess whether increased cytosolic Ca2+ originating from the ER is detrimental for neurons. Overexpression of IP3R2 in N2a cells using a Thy1.2–IP3R2 construct increases cytosolic Ca2+ concentrations evoked by bradykinin. In addition, mice generated from this construct have increased expression of IP3R2 in the spinal cord and brain. This overexpression of IP3R2 does not affect symptom onset, but decreases disease duration and shortens the lifespan of the ALS mice significantly. These data suggest that ER Ca2+ released by IP3 receptors may be detrimental in ALS and that motor neurons are vulnerable to impaired Ca2+ metabolism.

Highlights

► Overexpression of IP3R2 increases cytosolic Ca2+ concentrations evoked by bradykinin. ► Thy1.2–IP3R2 mice have increased expression of IP3R2 in the spinal cord and brain. ► Neuronal overexpression of IP3R2 shortens the lifespan of the ALS mice.

Introduction

A tight control of intracellular Ca2+ concentration is crucial for cell survival [1]. Motor neurons are especially vulnerable to disturbances in Ca2+ dynamics as they combine a low concentration of Ca2+ binding proteins with a relatively high amount of Ca2+-permeable AMPA-type glutamate receptors [2]. This could contribute to the selective vulnerability of the motor neurons during Amyotrophic Lateral Sclerosis (ALS). ALS is a progressive neurodegenerative disease, characterized by the selective loss of motor neurons and the denervation of muscle fibers, resulting in muscle weakness and paralysis. In Europe, the disease has an annual incidence of 2.7 cases per 100,000 people [3] and the disease duration post diagnosis is 3–5 years. In 10% of patients, ALS is a familial disease and 20% of these familial ALS patients contain mutations in the gene encoding superoxide dismutase 1 (SOD1). As the disease progression is indistinguishable between familial and sporadic ALS, common disease mechanisms are predicted. One of these mechanisms is excitotoxicity that causes neuronal death by overstimulation of the glutamate receptors [2]. The only effective treatment in ALS, riluzole, reduces the glutamatergic input [2]. In general, excitotoxicity induces an increased intracellular Ca2+ concentration, which is hazardous to the motor neurons. In addition, release of Ca2+ from the endoplasmic reticulum (ER) may further increase the intracellular Ca2+ concentration resulting in increased excitotoxicity [4].

In this study, we investigate the role of elevated expression of an ER Ca2+ release channel, inositol 1,4,5-trisphosphate receptor 2 (IP3R2) and concomitant increased IP3-induced ER Ca2+ release in ALS. To this end, we generated a mouse with a neuronal overexpression of IP3R2 and crossbred these mice with a mouse model for ALS, in which excitotoxicity contributes to motor neuron death [2].

Section snippets

Animal generation and housing

Mice overexpressing human SOD1G93A were purchased from The Jackson Laboratories (Bar Harbor, USA) and maintained on a C57BL/6 background. In order to create transgenic mice, murine itpr2 cDNA (8.1 kb) was cloned into the XhoI restriction site of the Thy1.2 expression cassette (kind gift from Novartis Pharma). A 15 kb DNA fragment was excised from the Thy1.2–IP3R2 vector with the restriction endonuclease Not1. The transgenic mice overexpressing itpr2 (Thy1.2–IP3R2) were created by pronuclear

IP3R2 overexpression increases agonist-induced Ca2+ signals in N2a cells

To investigate whether increased ER Ca2+ release through the IP3 receptor may be detrimental in ALS, we cloned the cDNA of the murine ITPR2 gene into the Thy1.2 expression cassette (Fig. 1A). The Thy1.2 expression cassette drives transgene expression starting at postnatal day 9 and especially in neurons [5]. IP3R2 is the most sensitive isoform of the receptor to IP3 [6], [7]. In the nervous system IP3R2 is mainly localized in astrocytes [8], [9], [10], [11], [12], [13]. The functional

Discussion

We studied the potential contribution of Ca2+ originating from intracellular stores and the role for ER Ca2+ release channels in neurons during ALS. A potential role of IP3R2 was previously suggested by genome wide association of single nucleotide polymorphisms (SNPs) in the ITPR2 gene with sporadic ALS [14], although this association was not found in other populations [15], [16]. Interestingly, an increase of IP3R2 gene expression is detected in blood samples of ALS patients [14]. Although IP3

Acknowledgments

This work was supported by grants from the “Fund for Scientific Research Flanders” (FWO-Vlaanderen, FWO G.0608.09N), the University of Leuven (KU Leuven, including GOA/11/014 and OTSTART1/10/044) and the Belgian Government (Interuniversity Attraction Poles, programme P7/16) of the Belgian Federal Science Policy Office. PVD holds a clinical investigatorship of FWO-Vlaanderen and WR is supported through the E. von Behring Chair for Neuromuscular and Neurodegenerative Disorders.

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