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Expression and regulation of FRMD6 in mouse DRG neurons and spinal cord after nerve injury.

ABSTRACT: FRMD6, a member of the group of FERM-domain proteins, is involved both in communication between cells, interactions with extracellular matrix, cellular apoptotic and regenerative mechanisms. FRMD6 was first discovered in the rodent sciatic nerve, and in the present immunohistochemical study we investigated the distribution of FRMD6 in the dorsal root ganglia (DRGs), sciatic nerve and spinal cord following sciatic nerve injury. FRMD6-immunoreactivity was found in the cytoplasm, nucleus or both, and in a majority of DRG neurons. FRMD6-immunoreactivity co-existed with several well-known neuronal markers, including calcitonin gene-related peptide, isolectin B4 and neurofilament 200 in mouse DRGs. After peripheral nerve injury, the FRMD6 mRNA levels and the overall percentage of FRMD6-positive neuron profiles (NPs) were decreased in ipsilateral lumbar DRGs, the latter mainly affecting small size neurons with cytoplasmic localization. Conversely, the proportion of NPs with nuclear FRMD6-immunoreactivity was significantly increased. In the sciatic nerve, FRMD6-immunoreactivity was observed in non-neuronal cells and in axons, and accumulated proximally to a ligation of the nerve. In the spinal cord FRMD6-immunoreactivity was detected in neurons in both dorsal and ventral horns, and was upregulated in ipsilateral dorsal horn after peripheral nerve axotomy. Our results demonstrate that FRMD6 is strictly regulated by peripheral nerve injury at the spinal level.

SUBMITTER: Lyu C

PROVIDER: S-EPMC7002571 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Expression and regulation of FRMD6 in mouse DRG neurons and spinal cord after nerve injury.

Lyu Chuang C Lyu Gong-Wei GW Mulder Jan J Uhlén Mathias M Cai Xue-Hui XH Hökfelt Tomas T Sten Shi Tie-Jun TJ

Scientific reports 20200205 1

FRMD6, a member of the group of FERM-domain proteins, is involved both in communication between cells, interactions with extracellular matrix, cellular apoptotic and regenerative mechanisms. FRMD6 was first discovered in the rodent sciatic nerve, and in the present immunohistochemical study we investigated the distribution of FRMD6 in the dorsal root ganglia (DRGs), sciatic nerve and spinal cord following sciatic nerve injury. FRMD6-immunoreactivity was found in the cytoplasm, nucleus or both, a ...[more]

PMID: 32024965

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Project description:Dysfunction of the fast-inactivating Kv3.4 potassium current in dorsal root ganglion (DRG) neurons contributes to the hyperexcitability associated with persistent pain induced by spinal cord injury (SCI). However, the underlying mechanism is not known. In light of our previous work demonstrating modulation of the Kv3.4 channel by phosphorylation, we investigated the role of the phosphatase calcineurin (CaN) using electrophysiological, molecular, and imaging approaches in adult female Sprague Dawley rats. Pharmacological inhibition of CaN in small-diameter DRG neurons slowed repolarization of the somatic action potential (AP) and attenuated the Kv3.4 current. Attenuated Kv3.4 currents also exhibited slowed inactivation. We observed similar effects on the recombinant Kv3.4 channel heterologously expressed in Chinese hamster ovary cells, supporting our findings in DRG neurons. Elucidating the molecular basis of these effects, mutation of four previously characterized serines within the Kv3.4 N-terminal inactivation domain eliminated the effects of CaN inhibition on the Kv3.4 current. SCI similarly induced concurrent Kv3.4 current attenuation and slowing of inactivation. Although there was little change in CaN expression and localization after injury, SCI induced upregulation of the native regulator of CaN 1 (RCAN1) in the DRG at the transcript and protein levels. Consistent with CaN inhibition resulting from RCAN1 upregulation, overexpression of RCAN1 in naive DRG neurons recapitulated the effects of pharmacological CaN inhibition on the Kv3.4 current and the AP. Overall, these results demonstrate a novel regulatory pathway that links CaN, RCAN1, and Kv3.4 in DRG neurons. Dysregulation of this pathway might underlie a peripheral mechanism of pain sensitization induced by SCI.SIGNIFICANCE STATEMENT Pain sensitization associated with spinal cord injury (SCI) involves poorly understood maladaptive modulation of neuronal excitability. Although central mechanisms have received significant attention, recent studies have identified peripheral nerve hyperexcitability as a driver of persistent pain signaling after SCI. However, the ion channels and signaling molecules responsible for this change in primary sensory neuron excitability are still not well defined. To address this problem, this study used complementary electrophysiological and molecular methods to determine how Kv3.4, a voltage-gated K+ channel robustly expressed in dorsal root ganglion neurons, becomes dysfunctional upon calcineurin (CaN) inhibition. The results strongly suggest that CaN inhibition underlies SCI-induced dysfunction of Kv3.4 and the associated excitability changes through upregulation of the native regulator of CaN 1 (RCAN1).

Project description:BACKGROUND: Spinal cord injury leads to neurological dysfunctions affecting the motor, sensory as well as the autonomic systems. Increased excitability of motor neurons has been implicated in injury-induced spasticity, where the reappearance of self-sustained plateau potentials in the absence of modulatory inputs from the brain correlates with the development of spasticity. RESULTS: Here we examine the dynamic transcriptional response of motor neurons to spinal cord injury as it evolves over time to unravel common gene expression patterns and their underlying regulatory mechanisms. For this we use a rat-tail-model with complete spinal cord transection causing injury-induced spasticity, where gene expression profiles are obtained from labeled motor neurons extracted with laser microdissection 0, 2, 7, 21 and 60 days post injury. Consensus clustering identifies 12 gene clusters with distinct time expression profiles. Analysis of these gene clusters identifies early immunological/inflammatory and late developmental responses as well as a regulation of genes relating to neuron excitability that support the development of motor neuron hyper-excitability and the reappearance of plateau potentials in the late phase of the injury response. Transcription factor motif analysis identifies differentially expressed transcription factors involved in the regulation of each gene cluster, shaping the expression of the identified biological processes and their associated genes underlying the changes in motor neuron excitability. CONCLUSIONS: This analysis provides important clues to the underlying mechanisms of transcriptional regulation responsible for the increased excitability observed in motor neurons in the late chronic phase of spinal cord injury suggesting alternative targets for treatment of spinal cord injury. Several transcription factors were identified as potential regulators of gene clusters containing elements related to motor neuron hyper-excitability, the manipulation of which potentially could be used to alter the transcriptional response to prevent the motor neurons from entering a state of hyper-excitability.

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Project description:BackgroundSpinal cord injury (SCI) induces a multitude of deleterious processes, including neuroinflammation and oxidative stress (OS) which contributed to neuronal damage and demyelination. Recent studies have suggested that increased formation of reactive oxygen species (ROS) and the consequent OS are critical events associated with SCI. However, there is still little information regarding the impact of these events on SCI. Astrocytes are key regulators of oxidative homeostasis in the CNS and astrocytic antioxidant responses promote the clearance of oxidants produced by neurons. Therefore, dysregulation of astrocyte physiology might largely contribute to oxidative damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the main transcriptional regulator of cellular anti-oxidative stress responses.MethodsIn the current study, we hypothesized that astrocytic activation of Nrf2 protects the spinal cord post injury via suppression of neuroinflammation. Thus, using mice line with a GFAP-specific kelch-like ECH-associated protein 1 (Keap1)-deletion, we induced a hyperactivation of Nrf2 in astrocytes and further its effects on SCI outcomes. SCI-induction was performed in mice using the Infinite Horizon Spinal Cord Impactor with a force of 60 kdyn. To assess the quantitative pattern of Nrf2/ARE-activation, we included transgenic ARE-Luc mice. Data were analyzed with GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Brown-Forsythe test was performed to test for equal variances and normal distribution was tested with Shapiro-Wilk.ResultsIn ARE-Luc mice, a significant induction of luciferase-activity was observed as early as 1 day post-injury, indicating a functional role of Nrf2-activity at the epicenter of SCI. Furthermore, SCI induced loss of neurons and oligodendrocytes, demyelination and inflammation in wild type mice. The loss of myelin and oligodendrocytes was clearly reduced in Keap1 KO mice. In addition, Keap-1 KO mice showed a significantly better locomotor function and lower neuroinflammation responses compared to wild type mice.ConclusionsIn summary, our in vivo bioluminescence data showed Nrf2-ARE activation during primary phase of SCI. Furthermore, we found that cell specific hyperactivation of Nrf2 was sufficient to protect the spinal cord against injury which indicate a promising therapeutic approach for SCI-treatment.

Dataset Information

Expression and regulation of FRMD6 in mouse DRG neurons and spinal cord after nerve injury.

Publications

Expression and regulation of FRMD6 in mouse DRG neurons and spinal cord after nerve injury.

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