Research in the Campana Lab
We study how peripheral glia, Schwann cells (SCs), affect neuropathic pain processes. We focus on the molecular and cellular response of nerve injury to better understand the mechanisms underlying the development, and transition to chronic pain.
Schwann cell dysfunction and neuropathic pain
While it is known that SCs are essential for successful peripheral nerve regeneration, the molecular mechanisms underlying these activities and how they influence pain states remains unclear. My lab has made fundamental discoveries identifying injury-activated receptors in SCs, including the erythropoietin receptor (EpoR), the low-density lipoprotein receptor related protein (LRP1) and the N-Methyl-D-Aspartate-Receptor (NMDA-R). This research established the foundation for a better understanding of SC function and the development of painful peripheral neuropathies. For example, erythropoietin, and its receptor, EpoR were thought to only exist in the hematological systems. However, we discovered that EpoR is localized in SCs, and that activation of SC EpoR was an important
anti-inflammatory pathway in injured peripheral nerves. One of the key underlying mechanisms was that Epo/EpoR activation facilitated the recruitment of B1-integrin to the SC surface, and thus, promoted SC adhesion and migration. Both anti-inflammatory and B1 integrin regulation by Epo/EpoR contribute to facilitated sensory nerve repair. Ultimately, these Epo/EpoR-induced activities correlated with a reduction in chronic pain. This innovative work led to a patent focused on erythropoietin as a potential therapeutic for painful peripheral neuropathies.
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Previously, the approach to pain management and drug development has been largely focused on sensory neurons, while targeting Schwann cells (SCs) has been largely underappreciated. Given the local intimacy of Schwann cells with sensory axons, a functional unit is formed that is essential and linked by multiple reciprocal signals that determine nerve health in development, in the normal adult, and following nerve injury (Campana, 2007, Brain Behav Immun 21:522-527). SCs provide myelination to large caliber axons, dramatically improving the speed and efficiency of action potential propagation.
Other SCs, in Remak bundles, do not generate myelin, but still provide metabolic and trophic support for axons. Remak bundle SCs ensheath small diameter axons (C-fibers) that are involved in pain transmission. Only recently, have studies using transgenic mouse models, in which SC physiology is disrupted, revealed important roles for SCs in pain chronicity. For example, we developed a novel transgenic mouse in which the gene (GRIN1) encoding the essential GluN1 NMDA-R subunit was deleted conditionally in SCs (scGRIN mice) (Brifault et al., 2020, J Neurosci 40(47):9121-9136).
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GluN1 is a bioactive receptor in SCs that is activated by glutamate and protein ligands (Campana et al., 2017, FASEB J 31: 1744-1755). In the absence of PNS injury, GRIN1 deletion in SCs caused hypersensitivity in pain-processing and significantly upregulated a “pain-associated” DRG transcriptome. These abnormalities were associated with changes in the density of small fibers in the skin and ultrastructural changes in Remak bundles. In adult scGRIN1 mice, Remak bundle ensheathment abnormalities were prevalent, resulting in highly variable spacing of neighboring axons (Fig. 1). This is significant because: 1) When C-fibers come into direct contact, they provide a mechanism for impulse conduction through the nerve membrane in either direction and spontaneous ephaptic transmission (Devor et al., 1993, J Neurosci 13:1976 –1993); and 2) When the distance between axons is abnormally large or variable, nerve firing also may become aberrant.
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Fig. 1. GRIN1 deletion in Schwann cells alters Remak bundles. Representative images of Remak bundles in GluN1+ (top panel) and GluN1- (bottom panel) sciatic nerves (4000x. Scale bar 1 µM)
Quantification of abnormalities in Remak bundles revealed significant differences in the genotypes. Abnormalities are defined as 1) SCs cytoplasm invagination (bottom left image); 2) poly-axonal pockets (yellow asterisk); and 3) axons in direct contact with the endonerium (pink arrowheads).
Other examples of SC dysfunction leading to aberrant sensory outcomes include changes in neuregulin NRG1 signaling. When the receptor for NRG1, Erb2/3, is conditionally deleted in SCs, robust tactile allodynia and eventual sensory loss are observed (Chen et al., 2003, Nat Neurosci 6:1186 –1193.).
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We pioneered studies with LRP1 in the peripheral nervous system (PNS), and specifically its role in SCs. Accordingly, we demonstrated that LRP1 is a major orchestrator of the SC Repair Program. Within hours after nerve injury, LRP1 is dramatically upregulated in SCs. LRP1 binds many endogenous ligands released into the injured nerve milieu, including proteases such as matrix metalloprotease-9 (MMP-9) and the intracellular neuronal protein, PACSIN-1. LRP1 couple’s receptor-mediated endocytosis with cell-signaling. Without LRP1 present in the immediate injured nerve environment, SCs are less likely to transdifferentiate into a SC repair phenotype, and nerve repair fails. Importantly, LRP1 ligands activate c-Jun, a key cell signaling protein in the SC Repair Program. LRP1 ligands such as PEX and tissue plasminogen activator (tPA) activate c-Jun in primary cultured SCs and in nerves 24 hours after injury.
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We developed a novel transgenic mouse line (a conditional knock out of LRP1 in SCs) to test the hypotheses that SC LRP1 is essential for functional peripheral nerve repair. When Lrp1 is conditionally deleted in SCs (scLRP1 ), mice show evidence of SC apoptosis after crush injury, suggesting that LRP1 is essential for SC survival. In addition, scLRP1 mice exhibit mechanical sensitivity prior to injury, and after injury have a greater magnitude and extent of neuropathic pain-related behaviors. Our findings supported an innovative hypothesis, that SC physiology contribute to pain states. This work was awarded F1000Prime recognition and a foundation for an important link between SC physiology and pain.
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Mitochondrial dynamics in peripheral nerves: effects of LRP1 on SC mitochondria
Painful peripheral neuropathies have heterogeneous etiologies and can emerge from traumatic, metabolic and chemotherapy induced events. There are no curative treatments for painful peripheral neuropathy (PPN); currently therapeutic options involve steroids, local anesthetics, anti-seizures drugs, and opioids. All treatments aim at temporarily reducing pain to a manageable level; however, several include clinically significant side effects and/or addiction. Thus, identifying novel strategies for improved pain treatment addresses this substantial unmet medical need. Mitochondria dysregulation has emerged as a mechanism associated with PPN (Boyette-Davis et al., 2018, Pain Manag 8(5):363-375). Yet, most of our understanding comes from neurons. Indeed, several studies have shown that in distal axons mitochondrial DNA mutations accumulate and contribute to PPN (Carelli & Chan, 2014 Neuron 84(6):1126-42), however, heterogeneities within SCs, is not known. Studies in which key mitochondrial proteins are genetically deleted in SCs, including mitochondrial transcription factor A gene (Tfam) (Viader et al., 2011, J Neurosci 31(28):10128- 40).or prohibitin (Della-Flora Nunes et al., 2021, Nat Commun 12(1):3285) demonstrate that abnormalities in SC mitochondrial function create sensory and motor deficits. These findings are consistent with data from human patients with PPN showing pathological mitochondria in SCs of neuropathic nerves (Kalichman et al., 1998, Acta Neuropathol 95(1):47-56). Currently, there are no studies linking a SC repair receptor signaling pathway (which could be targeted therapeutically) with changes in mitochondria heterogeneity/homeostasis relevant to neuropathic pain. Another important property of LRP1 is its ability to regulate lipid metabolism and glucose homeostasis, and therefore, control cellular bioenergetics. In hepatocytes, LRP1 directly regulates mitochondrial dynamics and function by reducing lipid kinase signaling (Chinnarasu, 2021, J Biol Chem 296:100370). Currently, studies are underway in my lab to determine whether LRP1 directly regulates mitochondrial dynamics and function in SCs to optimize bioenergetic homeostasis in peripheral nerves. In Fig. 2 we demonstrate mitochondrial heterogeneities in transgenic mice with genetic deletion of LRP1 in SCs. This work is an ongoing collaboration with Dr. Gulcin Pekkurnaz’s lab in Neurobiology.
Fig. 2. Mouse SCs (mSCs) cultured from adult scLRP1 and scLRP1 nerves. Representative IF images of primary cultured mSCs, stained with AntiPDH (green) and Dapi (blue). Arrows indicate the differences in mitochondrial morphology in soma and processes. Same image acquisition settings were used across genotypes.
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Schwann cell small extracellular vesicles as communicators in PNS homeostasis, in response to injury and established neuropathic pain.
Several studies have revealed that SCs release small extracellular vesicles (sEVs). Small EVs are small nanovesicles (40-150 nm) and include exosomes. Exosomes are derived from the endosomal system and contain nucleic acids, including non-coding microRNAs (miRs), and protein cargo. Initially, exosomes were thought to be a cellular mechanism of waste disposal but are now also considered to be highly specific enablers of cellular communication. Importantly, exosomes reflect the status of their originating cells. Our funded project investigates both healthy and neuropathic SC exosomes and how their distinct cargo regulates chronic pain states. We have established two innovative protocols to study SC-enriched exosomes in vivo. Plasma derived sEVs from healthy and neuropathic mice are isolated and enriched for SC sEVs by immunoprecipitation with a cell surface SC biomarker. We have also generated novel transgenic mice that have Cre inducible GFP-labeled CD9 that is specifically expressed in SCs and corresponding sEVs in vivo. We characterize SC EVs after isolation in vivo and in vitro by Nanoparticle Tracking Analyses (NTA) and EM (Fig. 3). We recently demonstrated a novel mechanism by which EVs produced by SCs may regulate cell physiology, involving the EV plasma membrane. SCs in primary culture produce EVs that are highly enriched in tumor necrosis factor receptor-1 (TNFR1) TNFR1 accounted for as much as 2% of the total protein of the SC EVs (Sadri et al., 2022 GLIA 70(2):256-272).
Fig. 3. Characterization of SC EVs. NTA (left) showing abundant small EVs and EM (right) showing EVs with crescent morphology.
SC EV-associated TNFR1 was biologically active, binding TNFα and preventing TNFα from initiating cell-signaling in cultured SCs in vitro and in vivo, when injected into sciatic nerves. We interpreted these results to indicate that SC EVs express TNFα “decoy activity” and may inhibit TNFα from engaging TNFα receptors in diverse cells in the injured PNS.
Fig. 4. Selective sequestration of TNFR1 into SC extracellular vesicles may regulate the response to PNS injury.
Furthermore, by purging TNFR1 from the SC, EV formation shifts the balance between TNFR1 and TNFR2 in the EV-generating cell in favor of TNFR2. This is important because TNFR2 lacks a death domain and thus, may function preferentially in tissue repair and regeneration. Our results suggest that SC EV formation may be critical in mediating transitions in SC phenotype required to support PNS regeneration, as opposed to Wallerian degeneration. Furthermore, by preventing TNFα from engaging receptors in macrophages in the injured PNS, SC EVs may facilitate a transition in macrophage populations from cells with M1-like properties to cells with M2-like properties. This transition in macrophage phenotype also may favor processes associated with tissue repair and regeneration. Pathways by which selective sequestration of TNFR1 into SC EVs may facilitate repair of the injured PNS are diagrammed in Fig. 4 (Gonias & Campana, 2023, Neural Regen Research 18(2): 325-326).