Adult Dorsal Root Ganglion Research Articles

Photopolymerized Microfeatures Guide Adult Spiral Ganglion and Dorsal Root Ganglion Neurite Growth.

Microtopographical patterns generated by photopolymerization of methacrylate polymer systems will direct growth of neurites from adult neurons, including spiral ganglion neurons (SGNs). Cochlear implants (CIs) provide hearing perception to patients with severe to profound hearing loss. However, their ability to encode complex auditory stimuli is limited due, in part, to poor spatial resolution caused by spread of the electrical currents in the inner ear. Directing the regrowth of SGN peripheral processes towards stimulating electrodes could help reduce current spread and improve spatial resolution provided by the CI. Previous work has demonstrated that micro- and nano-scale patterned surfaces precisely guide the growth of neurites from a variety of neonatal neurons including SGNs. Here, we sought to determine the extent to which adult neurons likewise respond to these topographical surface features. Photopolymerization was used to fabricate methacrylate polymer substrates with micropatterned surfaces of varying amplitudes and periodicities. Dissociated adult dorsal root ganglion neurons (DRGNs) and SGNs were cultured on these surfaces and the alignment of the neurite processes to the micropatterns was determined. Neurites from both adult DRGNs and SGNs significantly aligned to the patterned surfaces similar to their neonatal counterparts. Further DRGN and SGN neurite alignment increased as the amplitude of the microfeatures increased. Decreased pattern periodicity also improved neurite alignment. Microscale surface topographic features direct the growth of adult SGN neurites. Topographical features could prove useful for guiding growth of SGN peripheral axons towards a CI electrode array.

Neurons and satellite glial cells in adult rat lumbar dorsal root ganglia express connexin 36

Previous studies have shown that following peripheral nerve injury there was a downregulation of the gap junction protein connexin 36 (Cx36) in the spinal cord; however, it is not known whether Cx36 protein is expressed in the dorsal root ganglia (DRGs), nor if its levels are altered following peripheral nerve injuries. Here we address these aspects in the adult rat lumbar DRG. Cx36 mRNA was detected using qRT-PCR, and Cx36 protein was identified in DRG sections using immunohistochemistry (IHC) and immunofluorescence (IF). Double staining revealed that Cx36 co-localizes with both anti-β-III tubulin, a neuronal marker, and anti-glutamine synthetase, a satellite glial cell (SGC) marker. In neurons, Cx36 staining was mostly uniform in somata and fibers of all sizes and its intensity increased at the cell membranes. This labeling pattern was in contrast with Cx36 IF dots mainly found at junctional membranes in islet beta cells used as a control tissue. Co-staining with anti-Cx43 and anti-Cx36 showed that whereas mostly uniform staining of Cx36 was found throughout neurons and SGCs, Cx43 IF puncta were localized to SGCs. Cx36 mRNA was expressed in normal lumbar DRG, and it was significantly down-regulated in L4 DRG of rats that underwent sciatic nerve injury resulting in persistent hypersensitivity. Collectively, these findings demonstrated that neurons and SGCs express Cx36 protein in normal DRG, and suggested that perturbation of Cx36 levels may contribute to chronic neuropathic pain resulting from a peripheral nerve injury.

Trimethylene carbonate-caprolactone conduit with poly-p-dioxanone microfilaments to promote regeneration after spinal cord injury.

Biosynthetic conduits can support regeneration after spinal cord injury but often require addition of cell therapy and neurotrophic factors. This study demonstrates that biodegradable conduits made from trimethylene carbonate and ε-caprolactone with poly-p-dioxanone microfilaments alone can promote migration of different host cells and stimulate axonal regeneration after implantation into acute and chronic spinal cord injury. These results can be used to develop biosynthetic conduits for future clinical applications.

Antidepressant Imipramine Protects Bupivacaine‐Induced Neurotoxicity in Dorsal Root Ganglion Neurons Through Coactivation of TrkA and TrkB

In our work, we used an in vitro culture model to investigate whether antidepressant imipramine (Ip) may protect bupivacaine (Bv)-induced neurotoxicity in mouse dorsal root ganglion (DRG). Adult mouse DRG was treated with 5 mM Bv in vitro to induce neurotoxicity. DRG was then pre-treated with Ip, prior to Bv, to examine its effects on protecting Bv-induced DRG apoptosis and neurite degeneration. Ip-induced dynamic changes in Trk receptors, including TrkA/B/C and phosphor (p-)TrkA/B/C, were examined by qPCR and Western blot. TrkA and TrkB were inhibited by siRNAs to further investigate their functional role in Ip- and Bv-treated DRG. Ip protected Bv-induced apoptosis and neurite loss in DRG. Ip did not alter TrkA/B/C expressions, whereas significantly augmented protein productions of p-TrkA and p-TrkB, but not p-TrkC. SiRNA-mediated TrkA or TrkB downregulation inhibited Trk receptors, and reduced p-TrkA and p-TrkB in DRG. TrkA or TrkB downregulation alone had no effect on Ip-induced protection in Bv-injured DRG. However, co-inhibition of TrkA and TrkB significantly ameliorated the protective effect of Ip on Bv-induced apoptosis and neurite loss in DRG. Imipramine protected bupivacaine-induced neurotoxicity in DRG, likely via the co-activation of TrkA and TrkB signaling pathways. J. Cell. Biochem. 118: 3960-3967, 2017. © 2017 Wiley Periodicals, Inc.

Dyslipidemia impairs mitochondrial trafficking and function in sensory neurons.

Mitochondrial trafficking plays a central role in dorsal root ganglion (DRG) neuronal cell survival and neurotransmission by transporting mitochondria from the neuronal cell body throughout the bundles of DRG axons. In type 2 diabetes (T2DM), dyslipidemia and hyperglycemia damage DRG neurons and induce mitochondrial dysfunction; however, the impact of free fatty acids and glucose on mitochondrial trafficking in DRG neurons remains unknown. To evaluate the impact of free fatty acids compared to hyperglycemia on mitochondrial transport, primary adult mouse DRG neuron cultures were treated with physiologic concentrations of palmitate and glucose and assessed for alterations in mitochondrial trafficking, mitochondrial membrane potential, and mitochondrial bioenergetics. Palmitate treatment significantly reduced the number of motile mitochondria in DRG axons, but physiologic concentrations of glucose did not impair mitochondrial trafficking dynamics. Palmitate-treated DRG neurons also exhibited a reduction in mitochondrial velocity, and impaired mitochondrial trafficking correlated with mitochondrial depolarization in palmitate-treated DRG neurons. Finally, we found differential bioenergetic effects of palmitate and glucose on resting and energetically challenged mitochondria in DRG neurons. Together, these results suggest that palmitate induces DRG neuron mitochondrial depolarization, inhibiting axonal mitochondrial trafficking and altering mitochondrial bioenergetic capacity.-Rumora, A. E., Lentz, S. I., Hinder, L. M., Jackson, S. W., Valesano, A., Levinson, G. E., Feldman, E. L. Dyslipidemia impairs mitochondrial trafficking and function in sensory neurons.

Combining Constitutively Active Rheb Expression and Chondroitinase Promotes Functional Axonal Regeneration after Cervical Spinal Cord Injury

After spinal cord injury (SCI), severed axons in the adult mammalian CNS are unable to mount a robust regenerative response. In addition, the glial scar at the lesion site further restricts the regenerative potential of axons. We hypothesized that a combinatorial approach coincidentally targeting these obstacles would promote axonal regeneration. We combined (1) transplantation of a growth-permissive peripheral nerve graft (PNG) into an incomplete, cervical lesion cavity; (2) transduction of neurons rostral to the SCI site to express constitutively active Rheb (caRheb; a Ras homolog enriched in brain), a GTPase that directly activates the growth-promoting pathway mammalian target of rapamycin (mTOR) via AAV-caRheb injection; and (3) digestion of growth-inhibitory chondroitin sulfate proteoglycans within the glial scar at the distal PNG interface using the bacterial enzyme chondroitinase ABC (ChABC). We found that expressing caRheb in neurons post-SCI results in modestly yet significantly more axons regenerating across a ChABC-treated distal graft interface into caudal spinal cord than either treatment alone. Excitingly, we found that caRheb+ChABC treatment significantly potentiates the formation of synapses in the host spinal cord and improves the animals' ability to use the affected forelimb. Thus, this combination strategy enhances functional axonal regeneration following a cervical SCI.

Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia.

Limited knowledge from mechanistic studies on adult sensory neuronal activity was generated, to some extent, in recapitulated adult in vivo 3D microenvironment. To fill this gap there is a real need to better characterize the adult dorsal root ganglia (aDRG) organotypic cultures to make these in vitro systems exploitable for different approaches, ranging from basic neurobiology to regenerative therapies, to address the sensory nervous system in adult stage. We conducted a direct head-to-head comparison of aDRG and embryonic DRG (eDRG) organotypic culture focusing on axonal growth, neuropeptides expression and receptors tyrosine kinase (RTK) activation associated with neuronal survival, proliferation and differentiation. To identify alterations related to culture conditions, these parameters were also addressed in retrieved aDRG and eDRG and compared with organotypic cultures. Under similar neurotrophic stimulation, aDRG organotypic cultures displayed lower axonal outgrowth rate supported by reduced expression of growth associated protein-43 and high levels of RhoA and glycogen synthase kinase 3 beta mRNA transcripts. In addition, differential alteration in sensory neuropeptides expression, namely calcitonin gene-related peptide and substance P, was detected and was mainly pronounced at gene expression levels. Among 39 different RTK, five receptors from three RTK families were emphasized: tropomyosin receptor kinase A (TrkA), epidermal growth factor receptors (EGFR, ErbB2 and ErbB3) and platelet-derived growth factor receptor (PDGFR). Of note, except for EGFR, the phosphorylation of these receptors was dependent on DRG developmental stage and/or culture condition. In addition, EGFR and PDGFR displayed alterations in their cellular expression pattern in cultured DRG. Overall we provided valuable information particularly important when addressing in vitro the molecular mechanisms associated with development, maturation and regeneration of the sensory nervous system.

Stress Increases Peripheral Axon Growth and Regeneration through Glucocorticoid Receptor-Dependent Transcriptional Programs.

Stress and glucocorticoid (GC) release are common behavioral and hormonal responses to injury or disease. In the brain, stress/GCs can alter neuron structure and function leading to cognitive impairment. Stress and GCs also exacerbate pain, but whether a corresponding change occurs in structural plasticity of sensory neurons is unknown. Here, we show that in female mice (Mus musculus) basal GC receptor (Nr3c1, also known as GR) expression in dorsal root ganglion (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions (M. musculus). In response to stress or GCs, adult DRG neurite growth increases through mechanisms involving GR-dependent gene transcription. In vivo, prior exposure to an acute systemic stress increases peripheral nerve regeneration. These data have broad clinical implications and highlight the importance of stress and GCs as novel behavioral and circulating modifiers of neuronal plasticity.

MTOR regulates peripheral nerve response to tensile strain.

While excessive tensile strain can be detrimental to nerve function, strain can be a positive regulator of neuronal outgrowth. We used an in vivo rat model of sciatic nerve strain to investigate signaling mechanisms underlying peripheral nerve response to deformation. Nerves were deformed by 11% and did not demonstrate deficits in compound action potential latency or amplitude during or after 6 h of strain. As revealed by Western blotting, application of strain resulted in significant upregulation of mammalian target of rapamycin (mTOR) and S6 signaling in nerves, increased myelin basic protein (MBP) and β-actin levels, and increased phosphorylation of neurofilament subunit H (NF-H) compared with unstrained (sham) contralateral nerves (P < 0.05 for all comparisons, paired two-tailed t-test). Strain did not alter neuron-specific β3-tubulin or overall nerve tubulin levels compared with unstrained controls. Systemic rapamycin treatment, thought to selectively target mTOR complex 1 (mTORC1), suppressed mTOR/S6 signaling, reduced levels of MBP and overall tubulin, and decreased NF-H phosphorylation in nerves strained for 6 h, revealing a role for mTOR in increasing MBP expression and NF-H phosphorylation, and maintaining tubulin levels. Consistent with stretch-induced increases in MBP, immunolabeling revealed increased S6 signaling in Schwann cells of stretched nerves compared with unstretched nerves. In addition, application of strain to cultured adult dorsal root ganglion neurons showed an increase in axonal protein synthesis based on a puromycin incorporation assay, suggesting that neuronal translational pathways also respond to strain. This work has important implications for understanding mechanisms underlying nerve response to strain during development and regeneration.NEW & NOTEWORTHY Peripheral nerves experience tensile strain (stretch) during development and movement. Excessive strain impairs neuronal function, but moderate strains are accommodated by nerves and can promote neuronal growth; mechanisms underlying these phenomena are not well understood. We demonstrated that levels of several structural proteins increase following physiological levels of nerve strain and that expression of a subset of these proteins is regulated by mTOR. Our work has important implications for understanding nerve development and strain-based regenerative strategies.

Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons.

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2-/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth.SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2) is also expressed in DRG. We show here that it is a developmentally downregulated but dynamic component of KARs in these neurons, that it contributes to regulated neurite regrowth in adult neurons, and that it is increased in adult mice after nerve injury. Our data confirm Neto2 as a KAR auxiliary subunit and expand our knowledge of the molecular composition of KARs in nociceptive neurons, a key piece in understanding the mechanistic contribution of KAR signaling to pain-processing circuits.

Comprehensive mapping of 5-hydroxymethylcytosine epigenetic dynamics in axon regeneration

ABSTRACTIn contrast to central nervous system neurons, dorsal root ganglia (DRG) neurons can switch to a regenerative state after peripheral axotomy. In a screen for chromatin regulators of the regenerative responses in this conditioning lesion paradigm, we identified Tet methylcytosine dioxygenase 3 (Tet3) as upregulated in DRG neurons, along with increased 5-hydroxymethylcytosine (5hmC). We generated genome-wide 5hmC maps in adult DRG, which revealed that peripheral and central axotomy (leading to no regenerative effect) triggered differential 5hmC changes that are associated with distinct signaling pathways. 5hmC was altered in a large set of regeneration-associated genes (RAGs), including well-known RAGs, such as Atf3, Bdnf, and Smad1, that regulate axon growth potential of DRG neurons, thus supporting its role for RAG regulation. Our analyses also predicted HIF-1, STAT, and IRF as potential transcription factors that may collaborate with Tet3 for 5hmC modifications. Intriguingly, central axotomy resulted in widespread 5hmC modifications that had little overlap with those of peripheral axotomy, thus potentially constituting a roadblock for regeneration. Our study revealed 5hmC dynamics as a previously unrecognized epigenetic mechanism underlying the divergent responses after axonal injury.

Dynamic Expression of Serotonin Receptor 5-HT3A in Developing Sensory Innervation of the Lower Urinary Tract.

Sensory afferent signaling is required for normal function of the lower urinary tract (LUT). Despite the wide prevalence of bladder dysfunction and pelvic pain syndromes, few effective treatment options are available. Serotonin receptor 5-HT3A is a known mediator of visceral afferent signaling and has been implicated in bladder function. However, basic expression patterns for this gene and others among developing bladder sensory afferents that could be used to inform regenerative efforts aimed at treating deficiencies in pelvic innervation are lacking. To gain greater insight into the molecular characteristics of bladder sensory innervation, we conducted a thorough characterization of Htr3a expression in developing and adult bladder-projecting lumbosacral dorsal root ganglia (DRG) neurons. Using a transgenic Htr3a-EGFP reporter mouse line, we identified 5-HT3A expression at 10 days post coitus (dpc) in neural crest derivatives and in 12 dpc lumbosacral DRG. Using immunohistochemical co-localization we observed Htr3a-EGFP expression in developing lumbosacral DRG that partially coincides with neuropeptides CGRP and Substance P and capsaicin receptor TRPV1. A majority of Htr3a-EGFP+ DRG neurons also express a marker of myelinated Aδ neurons, NF200. There was no co-localization of 5-HT3A with the TRPV4 receptor. We employed retrograde tracing in adult Htr3a-EGFP mice to quantify the contribution of 5-HT3A+ DRG neurons to bladder afferent innervation. We found that 5-HT3A is expressed in a substantial proportion of retrograde traced DRG neurons in both rostral (L1, L2) and caudal (L6, S1) axial levels that supply bladder innervation. Most bladder-projecting Htr3a-EGFP+ neurons that co-express CGRP, Substance P, or TRPV1 are found in L1, L2 DRG, whereas Htr3a-EGFP+, NF200+ bladder-projecting neurons are from the L6, S1 axial levels. Our findings contribute much needed information regarding the development of LUT innervation and highlight the 5-HT3A serotonin receptor as a candidate for future studies of neurally mediated bladder control.

Serine residues at positions 162 and 166 of the rabies virus phosphoprotein are critical for the induction of oxidative stress in rabies virus infection.

Our previous work in a mouse model of experimental rabies showed neuronal process (dendrites and axons) degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion (DRG) neurons infected with the challenge virus standard-11 (CVS) strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases mitochondrial complex I activity and reactive oxygen species (ROS) production. Expression of a peptide from amino acid 139-172 of the CVS phosphoprotein (P) increased complex I activity and ROS generation similar to expression of the entire P. Site-directed mutational analyses illustrated the importance of the 145-151 and 157-169 regions of P and that serine residues at 162 and 166 are important single amino acid sites. Two CVS recombinant viruses with serine to alanine mutations at positions 162 (A162r) and 166 (A166r) did not increase complex I activity or ROS generation and also did not induce axonal swellings or inhibit axonal growth in DRG neurons. RABV infection is a mitochondrial disorder initiated by interaction of the RABV P and complex I; S162 and S166 are critical sites in the P for this interaction. The resulting mitochondrial dysfunction produces oxidative stress in neurons causing acute degenerative changes affecting neuronal processes resulting in a severe and fatal clinical disease. This information will be important for the future development of novel therapies for rabies.

Characterization of dorsal root ganglion neurons cultured on silicon micro-pillar substrates.

Our study focuses on characterization of dorsal root ganglion (DRG) neurons cultured on silicon micro-pillar substrates (MPS) with the ultimate goal of designing micro-electrode arrays (MEAs) for successful electrophysiological recordings of DRG neurons. Adult and neonatal DRG neurons were cultured on MPS and glass coverslips for 7 days in vitro. DRG neuronal distribution and morphometric analysis, including neurite alignment and length, was performed on MPS areas with different pillar width and spacing. We showed that MPS provide an environment for growth of adult and neonatal DRG neurons as permissive as control glass surfaces. Neonatal DRG neurons were present on MPS areas with narrow pillar spacing, while adult neurons preferred wider pillar spacing. Compared to the control glass surfaces the neonatal and adult DRG neurons in regions with narrow pillar spacing range developed a smaller number of longer neurites. In the same area, neurites were preferentially oriented along three directional axes at 30°, 90° and 150°. MPS architecture influenced growth directionality of all main DRG neuronal subtypes. We can conclude that specific micro-pillar substrate topography affects the morphology of DRG neurons. This knowledge can enable development of MEAs with precisely defined physical features for various neuroscience applications.

Neurotoxic mechanisms of paclitaxel are local to the distal axon and independent of transport defects

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of paclitaxel and other chemotherapeutic agents. Paclitaxel binds and stabilizes microtubules, but the cellular mechanisms that underlie paclitaxel's neurotoxic effects are not well understood. We therefore used primary cultures of adult murine dorsal root ganglion neurons, the cell type affected in patients, to examine leading hypotheses to explain paclitaxel neurotoxicity. We address the role of microtubule hyperstabilization and its downstream effects. Paclitaxel administered at 10–50nM for 1–3days induced retraction bulbs at the tips of axons and arrested axon growth without triggering axon fragmentation or cell death. By correlating the toxic effects and microtubule stabilizing activity of structurally different microtubule stabilizing compounds, we confirmed that microtubule hyperstabilization, rather than an off-target effect, is the likely primary cause of paclitaxel neurotoxicity. We examined potential downstream consequences of microtubule hyperstabilization and found that changes in levels of tubulin posttranslational modifications, although present after paclitaxel exposure, are not implicated in the paclitaxel neurotoxicity we observed in the cultures. Additionally, defects in axonal transport were not implicated as an early, causative mechanism of paclitaxel's toxic effects on dorsal root ganglion neurons. By using microfluidic chambers to selectively treat different parts of the axon with paclitaxel, we found that the distal axon was primarily vulnerable to paclitaxel, indicating that paclitaxel acts directly on the distal axon to induce degenerative effects. Together, our findings point to local effects of microtubule hyperstabilization on the distal-most portion of the axon as an early mediator of paclitaxel neurotoxicity. Because sensory neurons have a unique and ongoing requirement for distal growth in order to reinnervate the epidermis as it turns over, we propose that the ability of paclitaxel to arrest their growth accounts for the selective vulnerability of sensory neurons to paclitaxel neurotoxicity.

Toxicological effect of inhaled mosquito coil smoke on male fertility: Oxidative stress and antioxidant response

Evidence is emerging for a role of rough endoplasmic reticulum (RER) in the form of stress granules, the unfolded protein response and protein bodies in the response of neurons to injury and in neurodegenerative diseases. Here, we have studied the role of the peripheral target in regulating the RER and polyribosomes of Nissl bodies in axotomised adult cat dorsal root ganglion (DRG) neurons where axonal regeneration and peripheral target reinnervation was either allowed or denied. Retrograde labelling with horseradish peroxidise was used as an independent marker to enable selection of only those DRG neuronal cell bodies with axons in the injured intercostal nerves. Indications of polyribosomal dispersal were seen by 6 h following axotomy, and by 24 h the normal orderly arrangement of lamellae of RER in Nissl bodies had become disorganised. These ultrastructural changes preceded light microscopical chromatolysis by 1–3 d. The retrograde response was maximal 8–32 d after axotomy. Clusters of debris-laden satellite cells/macrophages were present at this time but no ultrastructural evidence of neuronal apoptosis or necrosis was seen and there were no differences in the initial retrograde response according to the type of injury. By 64 d following axotomy with reinnervation, approximately half the labelled DRG neurons showed restoration of the orderly arrangement of RER and polyribosomes in their Nissl bodies. This was not seen after axotomy with reinnervation denied. We propose that the target-dependent changes in Nissl body ultrastructure described here are part of a continuum that can modify neuronal protein synthesis directed towards growth, maintenance or death of the neuron. This represents a possible structural basis for mediating the varied effects of neurotrophic interactions.

MiR-155 Deletion in Mice Overcomes Neuron-Intrinsic and Neuron-Extrinsic Barriers to Spinal Cord Repair.

Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers including inflammation. microRNA (miR)-155-5p is a small, noncoding RNA that negatively regulates mRNA translation. In macrophages, miR-155-5p is induced by inflammatory stimuli and elicits a response that could be toxic after SCI. miR-155 may also independently alter expression of genes that regulate axon growth in neurons. Here, we hypothesized that miR-155 deletion would simultaneously improve axon growth and reduce neuroinflammation after SCI by acting on both neurons and macrophages. New data show that miR-155 deletion attenuates inflammatory signaling in macrophages, reduces macrophage-mediated neuron toxicity, and increases macrophage-elicited axon growth by ∼40% relative to control conditions. In addition, miR-155 deletion increases spontaneous axon growth from neurons; adult miR-155 KO dorsal root ganglion (DRG) neurons extend 44% longer neurites than WT neurons. In vivo, miR-155 deletion augments conditioning lesion-induced intraneuronal expression of SPRR1A, a regeneration-associated gene; ∼50% more injured KO DRG neurons expressed SPRR1A versus WT neurons. After dorsal column SCI, miR-155 KO mouse spinal cord has reduced neuroinflammation and increased peripheral conditioning-lesion-enhanced axon regeneration beyond the epicenter. Finally, in a model of spinal contusion injury, miR-155 deletion improves locomotor function at postinjury times corresponding with the arrival and maximal appearance of activated intraspinal macrophages. In miR-155 KO mice, improved locomotor function is associated with smaller contusion lesions and decreased accumulation of inflammatory macrophages. Collectively, these data indicate that miR-155 is a novel therapeutic target capable of simultaneously overcoming neuron-intrinsic and neuron-extrinsic barriers to repair after SCI. Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers, including inflammation. Here, new data show that deleting microRNA-155 (miR-155) affects both mechanisms and improves repair and functional recovery after SCI. Macrophages lacking miR-155 have altered inflammatory capacity, which enhances neuron survival and axon growth of cocultured neurons. In addition, independent of macrophages, adult miR-155 KO neurons show enhanced spontaneous axon growth. Using either spinal cord dorsal column crush or contusion injury models, miR-155 deletion improves indices of repair and recovery. Therefore, miR-155 has a dual role in regulating spinal cord repair and may be a novel therapeutic target for SCI and other CNS pathologies.

Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

Axon injury in the peripheral nervous system (PNS) induces a regeneration-associated gene (RAG) response. Atf3 (activating transcription factor 3) is such a RAG and ATF3's transcriptional activity might induce ‘effector’ RAGs (e.g. small proline rich protein 1a (Sprr1a), Galanin (Gal), growth-associated protein 43 (Gap43)) facilitating peripheral axon regeneration. We provide a first analysis of Atf3 mouse mutants in peripheral nerve regeneration. In Atf3 mutant mice, facial nerve regeneration and neurite outgrowth of adult ATF3-deficient primary dorsal root ganglia neurons was decreased. Using genome-wide transcriptomics, we identified a neuropeptide-encoding RAG cluster (vasoactive intestinal peptide (Vip), Ngf, Grp, Gal, Pacap) regulated by ATF3. Exogenous administration of neuropeptides enhanced neurite growth of Atf3 mutant mice suggesting that these molecules might be effector RAGs of ATF3's pro-regenerative function. In addition to the induction of growth-promoting molecules, we present data that ATF3 suppresses growth-inhibiting molecules such as chemokine (C-C motif) ligand 2. In summary, we show a pro-regenerative ATF3 function during PNS nerve regeneration involving transcriptional activation of a neuropeptide-encoding RAG cluster. ATF3 is a general injury-inducible factor, therefore ATF3-mediated mechanisms identified herein might apply to other cell and injury types.

Expressing Constitutively Active Rheb in Adult Dorsal Root Ganglion Neurons Enhances the Integration of Sensory Axons that Regenerate Across a Chondroitinase-Treated Dorsal Root Entry Zone Following Dorsal Root Crush.

While the peripheral branch of dorsal root ganglion neurons (DRG) can successfully regenerate after injury, lesioned central branch axons fail to regrow across the dorsal root entry zone (DREZ), the interface between the dorsal root and the spinal cord. This lack of regeneration is due to the limited regenerative capacity of adult sensory axons and the growth-inhibitory environment at the DREZ, which is similar to that found in the glial scar after a central nervous system (CNS) injury. We hypothesized that transduction of adult DRG neurons using adeno-associated virus (AAV) to express a constitutively-active form of the GTPase Rheb (caRheb) will increase their intrinsic growth potential after a dorsal root crush. Additionally, we posited that if we combined that approach with digestion of upregulated chondroitin sulfate proteoglycans (CSPG) at the DREZ with chondroitinase ABC (ChABC), we would promote regeneration of sensory axons across the DREZ into the spinal cord. We first assessed if this strategy promotes neuritic growth in an in vitro model of the glial scar containing CSPG. ChABC allowed for some regeneration across the once potently inhibitory substrate. Combining ChABC treatment with expression of caRheb in DRG significantly improved this growth. We then determined if this combination strategy also enhanced regeneration through the DREZ after dorsal root crush in adult rats in vivo. After unilaterally crushing C4-T1 dorsal roots, we injected AAV5-caRheb or AAV5-GFP into the ipsilateral C5-C8 DRGs. ChABC or PBS was injected into the ipsilateral dorsal horn at C5-C8 to digest CSPG, for a total of four animal groups (caRheb + ChABC, caRheb + PBS, GFP + ChABC, GFP + PBS). Regeneration was rarely observed in PBS-treated animals, whereas short-distance regrowth across the DREZ was observed in ChABC-treated animals. No difference in axon number or length between the ChABC groups was observed, which may be related to intraganglionic inflammation induced by the injection. ChABC-mediated regeneration is functional, as stimulation of ipsilateral median and ulnar nerves induced neuronal c-Fos expression in deafferented dorsal horn in both ChABC groups. Interestingly, caRheb + ChABC animals had significantly more c-Fos+ nuclei indicating that caRheb expression in DRGs promoted functional synaptogenesis of their axons that regenerated beyond a ChABC-treated DREZ.

Group II mGluRs suppress hyperexcitability in mouse and human nociceptors.

We introduce a strategy for preclinical research wherein promising targets for analgesia are tested in rodent and subsequently validated in human sensory neurons. We evaluate group II metabotropic glutamate receptors, the activation of which is efficacious in rodent models of pain. Immunohistochemical analysis showed positive immunoreactivity for mGlu2 in rodent dorsal root ganglia (DRG), peripheral fibers in skin, and central labeling in the spinal dorsal horn. We also found mGlu2-positive immunoreactivity in human neonatal and adult DRG. RNA-seq analysis of mouse and human DRG revealed a comparative expression profile between species for group II mGluRs and for opioid receptors. In rodent sensory neurons under basal conditions, activation of group II mGluRs with a selective group II agonist produced no changes to membrane excitability. However, membrane hyperexcitability in sensory neurons exposed to the inflammatory mediator prostaglandin E2 (PGE2) was prevented by (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC). In human sensory neurons from donors without a history of chronic pain, we show that PGE2 produced hyperexcitability that was similarly blocked by group II mGluR activation. These results reveal a mechanism for peripheral analgesia likely shared by mice and humans and demonstrate a translational research strategy to improve preclinical validation of novel analgesics using cultured human sensory neurons.