Differential expression of ryanodine receptor isoforms after spinal cord injury

Highlights

• RyR2 and RyR3 are upregulated in dorsal root ganglion neurons acutely after SCI.

• RyR2 and RyR3 are upregulated in the lesion site after SCI.

• RyR1 remained unchanged acutely following SCI.

• RyR2 and RyR3 spatially overlap with dystrophic axons after SCI.

Abstract

Ryanodine receptors (RyRs) are highly conductive intracellular Ca²⁺ release channels and are widely expressed in many tissues, including the central nervous system. RyRs have been implicated in intracellular Ca²⁺ overload which can drive secondary damage following traumatic injury to the spinal cord (SCI), but the spatiotemporal expression of the three isoforms of RyRs (RyR1-3) after SCI remains unknown. Here, we analyzed the gene and protein expression of RyR isoforms in the murine lumbar dorsal root ganglion (DRG) and the spinal cord lesion site at 1, 2 and 7 d after a mild contusion SCI. Quantitative RT PCR analysis revealed that RyR3 was significantly increased in lumbar DRGs and at the lesion site at 1 and 2 d post contusion compared to sham (laminectomy only) controls. Additionally, RyR2 expression was increased at 1 d post injury within the lesion site. RyR2 and -3 protein expression was localized to lumbar DRG neurons and their spinal projections within the lesion site acutely after SCI. In contrast, RyR1 expression within the DRG and lesion site remained unaltered following trauma. Our study shows that SCI initiates acute differential expression of RyR isoforms in DRG and spinal cord.

Introduction

Spinal cord injury (SCI) inflicts damage to ascending and descending axons and thereby contributes to neurological impairment. Although the precise mechanisms of secondary axonal injury are not completely understood, intra-axonal Ca²⁺ overload is thought to play a major role [1], [2], [3]. Extracellular sources of Ca²⁺ influx in axons include voltage-gated Ca²⁺ [4], [5], [6], [7], transmitter-operated channels [8], [9], [10], [11], reversal of Na⁺-Ca²⁺ exchange [12], and diffusion through mechanopores [13]. However, most attempts to target extracellular-mediated Ca²⁺ entry in animal models of SCI [14], [15], [16], [17], [18] and clinical trials have failed [19]. Alternatively, other sources of pathological Ca²⁺ release may play an important role in axonal loss following SCI, as has been shown for ischemic axons [7].

Indeed, major intracellular sources of Ca²⁺ implicated in axonal injury include mitochondria [20], [21], and the ER (or axoplasmic reticulum in axons) [7], [22], [23], [24]. With regards to the latter, Ca²⁺ can be mobilized into the cytoplasm from intracellular Ca²⁺ stores by two main types of receptors through differential signaling pathways: RyRs and inositol 1,4,5-trisphosphate receptor (IP₃R). RyR exist in three isoforms in mammals (RyR1-3) and RyR1 and RyR2 are well known for their role in mobilizing Ca²⁺ during excitation-contraction coupling in skeletal and cardiac muscle respectively [25], [26]. All three RyR isoforms are differentially expressed in the CNS and have been implicated in diverse functions such as synaptic plasticity, vesicle fusion and synaptic release of transmitters, pain, and growth cone dynamics [27], [28], [29], [30], [31].

Although it is well established that Ca²⁺ toxicity occurs in neurons in several pathological disorders, and Ca²⁺ release from intracellular stores is clearly involved in these processes [32], [33], [34], [35], [22], [23], [36], [37], little is known about the role and potential expression changes of the different RyRs isoforms after SCI. One purpose of this study was to establish whether different time points after injury result in a differential transcriptional response of RyRs. Accordingly, in the present work we examined the gene and protein expression of RyRs in DRGs and spinal cord of mice at 1, 2 and 7 d after a contusion-induced SCI.