Ischemic post-conditioning attenuates acute lung injury induced by intestinal ischemia-reperfusion in mice: role of Nrf2.

Intestinal ischemic post-conditioning (IPo) protects against lung injury induced by intestinal ischemia-reperfusion (IIR) partly through promotion of expression and function of heme oxygenase-1 (HO-1). NF-E2-related factor-2 (Nrf2) is a key transcription factor that interacts with HO-1 and regulates antioxidant defense. However, the role of Nrf2 in IPo protection of IIR-induced pulmonary injury is not completely understood. Here we show that IPo significantly attenuated IIR-induced lung injury and suppressed oxidative stress and systemic inflammatory responses. IPo also increased the expression of both Nrf2 and HO-1. Consistently, the beneficial effects of IPo were abolished by ATRA and Brusatol, potent inhibitors of Nrf2. Moreover, the Nrf2 agonist t-BHQ showed similar activity as IPo. Taken together, our data suggest that Nrf2 activity, along with HO-1, plays an important role in the protective effects of IPo against IIR-induced acute lung injury.

Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord.

There are substantial disagreements about the expression of gastrin-releasing peptide (GRP) in sensory neurons and whether GRP antibody cross-reacts with substance P (SP). These concerns necessitate a critical revaluation of GRP expression using additional approaches. Here, we show that a widely used GRP antibody specifically recognizes GRP but not SP. In the spinal cord of mice lacking SP (Tac1KO), the expression of not only GRP but also other peptides, notably neuropeptide Y (NPY), is significantly diminished. We detectedGrpmRNA in dorsal root ganglias using reverse transcription polymerase chain reaction, in situ hybridization and RNA-seq. We demonstrated thatGrpmRNA and protein are upregulated in dorsal root ganglias, but not in the spinal cord, of mice with chronic itch. Few GRP(+)immunostaining signals were detected in spinal sections following dorsal rhizotomy and GRP(+)cell bodies were not detected in dissociated dorsal horn neurons. Ultrastructural analysis further shows that substantially more GRPergic fibers form synaptic contacts with gastrin releasing peptide receptor-positive (GRPR(+)) neurons than SPergic fibers. Our comprehensive study demonstrates that a majority of GRPergic fibers are of primary afferent origin. A number of factors such as low copy number ofGrptranscripts, small percentage of cells expressingGrp, and the use of an eGFP GENSAT transgenic as a surrogate for GRP protein have contributed to the controversy. Optimization of experimental procedures facilitates the specific detection of GRP expression in dorsal root ganglia neurons.

Cross-inhibition of NMBR and GRPR signaling maintains normal histaminergic itch transmission.

We previously showed that gastrin-releasing peptide receptor (GRPR) in the spinal cord is important for mediating nonhistaminergic itch. Neuromedin B receptor (NMBR), the second member of the mammalian bombesin receptor family, is expressed in a largely nonoverlapping pattern with GRPR in the superficial spinal cord, and its role in itch transmission remains unclear. Here, we report that Nmbr knock-out (KO) mice exhibited normal scratching behavior in response to intradermal injection of pruritogens. However, mice lacking both Nmbr and Grpr (DKO mice) showed significant deficits in histaminergic itch. In contrast, the chloroquine (CQ)-evoked scratching behavior of DKO mice is not further reduced compared with Grpr KO mice. These results suggest that NMBR and GRPR could compensate for the loss of each other to maintain normal histamine-evoked itch, whereas GRPR is exclusively required for CQ-evoked scratching behavior. Interestingly, GRPR activity is enhanced in Nmbr KO mice despite the lack of upregulation of Grpr expression; so is NMBR in Grpr KO mice. We found that NMB acts exclusively through NMBR for itch transmission, whereas GRP can signal through both receptors, albeit to NMBR to a much lesser extent. Although NMBR and NMBR(+) neurons are dispensable for histaminergic itch, GRPR(+) neurons are likely to act downstream of NMBR(+) neurons to integrate NMB-NMBR-encoded histaminergic itch information in normal physiological conditions. Together, we define the respective function of NMBR and GRPR in itch transmission, and reveal an unexpected relationship not only between the two receptors but also between the two populations of interneurons in itch signaling.

Axonal transport: how high microtubule density can compensate for boundary effects in small-caliber axons.

Long-distance intracellular axonal transport is predominantly microtubule-based, and its impairment is linked to neurodegeneration. In this study, we present theoretical arguments that suggest that near the axon boundaries (walls), the effective viscosity can become large enough to impede cargo transport in small (but not large) caliber axons. Our theoretical analysis suggests that this opposition to motion increases rapidly as the cargo approaches the wall. We find that having parallel microtubules close enough together to enable a cargo to simultaneously engage motors on more than one microtubule dramatically enhances motor activity, and thus minimizes the effects of any opposition to transport. Even if microtubules are randomly placed in axons, we find that the higher density of microtubules found in small-caliber axons increases the probability of having parallel microtubules close enough that they can be used simultaneously by motors on a cargo. The boundary effect is not a factor in transport in large-caliber axons where the microtubule density is lower.

B-type natriuretic peptide is neither itch-specific nor functions upstream of the GRP-GRPR signaling pathway.

BACKGROUND: A recent study by Mishra and Hoon identified B-type natriuretic peptide (BNP) as an important peptide for itch transmission and proposed that BNP activates spinal natriuretic peptide receptor-A (NPRA) expressing neurons, which release gastrin releasing peptide (GRP) to activate GRP receptor (GRPR) expressing neurons to relay itch information from the periphery to the brain (Science 340:968-971, 2013). A central premise for the validity of this novel pathway is the absence of GRP in the dorsal root ganglion (DRG) neurons. To this end, they showed that Grp mRNA in DRG neurons is either absent or barely detectable and claimed that BNP but not GRP is a major neurotransmitter for itch in pruriceptors. They showed that NPRA immunostaining is perfectly co-localized with Grp-eGFP in the spinal cord, and a few acute pain behaviors in Nppb-/- mice were tested. They claimed that BNP is an itch-selective peptide that acts as the first station of a dedicated neuronal pathway comprising a GRP-GRPR cascade for itch. However, our studies, along with the others, do not support their claims. FINDINGS: We were unable to reproduce the immunostaining of BNP and NPRA as shown by Mishra and Hoon. By contrast, we were able to detect Grp mRNA in DRGs using in situ hybridization and real time RT-PCR. We show that the expression pattern of Grp mRNA is comparable to that of GRP protein in DRGs. Pharmacological and genetic blockade of GRP-GRPR signaling does not significantly affect intrathecal BNP-induced scratching behavior. We show that BNP inhibits inflammatory pain and morphine analgesia. CONCLUSIONS: Accumulating evidence demonstrates that GRP is a key neurotransmitter in pruriceptors for mediating histamine-independent itch. BNP-NPRA signaling is involved in both itch and pain and does not function upstream of the GRP-GRPR dedicated neuronal pathway. The site of BNP action in itch and pain and its relationship with GRP remain to be clarified.

Expansion of neurofilament medium C terminus increases axonal diameter independent of increases in conduction velocity or myelin thickness.

Maturation of the peripheral nervous system requires specification of axonal diameter, which, in turn, has a significant influence on nerve conduction velocity. Radial axonal growth initiates with myelination, and is dependent upon the C terminus of neurofilament medium (NF-M). Molecular phylogenetic analysis in mammals suggested that expanded NF-M C termini correlated with larger-diameter axons. We used gene targeting and computational modeling to test this new hypothesis. Increasing the length of NF-M C terminus in mice increased diameter of motor axons without altering neurofilament subunit stoichiometry. Computational modeling predicted that an expanded NF-M C terminus extended farther from the neurofilament core independent of lysine-serine-proline (KSP) phosphorylation. However, expansion of NF-M C terminus did not affect the distance between adjacent neurofilaments. Increased axonal diameter did not increase conduction velocity, possibly due to a failure to increase myelin thickness by the same proportion. Failure of myelin to compensate for larger axonal diameters suggested a lack of plasticity during the processes of myelination and radial axonal growth.

Muscle pathology without severe nerve pathology in a new mouse model of Charcot-Marie-Tooth disease type 2E.

Mutations in neurofilament light (NF-L) have been linked to Charcot-Marie-Tooth disease type 2E (CMT2E) in humans. To provide insight into disease pathogenesis, we developed a novel line of CMT2E mice that constitutively express human NF-L (hNF-L) with a glutamic acid to lysine mutation at position 397 (hNF-L(E397K)). This new line of mice developed signs consistent with CMT2E patients. Disease signs were first observed at 4 months in hNF-L(E397K) mice, and consisted of aberrant hind limb posture, digit deformities, reduced voluntary locomotor activity, reduced motor nerve conduction velocities (MNCVs) and muscle atrophy. Reduced voluntary locomotor activity and muscle pathology occurred without significant denervation, and hNF-L(E397K) mice showed relatively mild signs of nerve pathology. Nerve pathology in hNF-L(E397K) mice was characterized by ectopic accumulations of phosphorylated NFs in motor neuron cell bodies as early as 1 month. Moreover, NF organization was altered in motor and sensory roots, with small motor axons being most affected. Peak axonal diameter was reduced for small motor axons prior to and after the onset of overt phenotypes, whereas large motor axons were affected only after onset, which correlated with reduced MNCVs. Additionally, there was a small reduction in the number of sensory axons in symptomatic hNF-L(E397K) mice. hNF-L(E397K) mice are a novel line of CMT2E mice that recapitulate many of the overt phenotypes observed in CMT2E patients and hNF-L(P22S) mice. The cellular pathology observed in hNF-L(E397K) mice differed from that recently reported in hNF-L(P22S) mice, suggesting that overt CMT2E phenotypes may arise through different cellular mechanisms.

A non-canonical retina-ipRGCs-SCN-PVT visual pathway for mediating contagious itch behavior.

Contagious itch behavior informs conspecifics of adverse environment and is crucial for the survival of social animals. Gastrin-releasing peptide (GRP) and its receptor (GRPR) in the suprachiasmatic nucleus (SCN) of the hypothalamus mediates contagious itch behavior in mice. Here, we show that intrinsically photosensitive retina ganglion cells (ipRGCs) convey visual itch information, independently of melanopsin, from the retina to GRP neurons via PACAP-PAC1R signaling. Moreover, GRPR neurons relay itch information to the paraventricular nucleus of the thalamus (PVT). Surprisingly, neither the visual cortex nor superior colliculus is involved in contagious itch. In vivo calcium imaging and extracellular recordings reveal contagious itch-specific neural dynamics of GRPR neurons. Thus, we propose that the retina-ipRGC-SCN-PVT pathway constitutes a previously unknown visual pathway that probably evolved for motion vision that encodes salient environmental cues and enables animals to imitate behaviors of conspecifics as an anticipatory mechanism to cope with adverse conditions.

The spinal muscular atrophy mouse model, SMAΔ7, displays altered axonal transport without global neurofilament alterations.

Spinal muscular atrophy (SMA) is a neurodegenerative disease resulting from decreased levels of survival motor neuron 1 (SMN1) protein. Reduced SMN1 levels are linked to pathology at neuromuscular junctions (NMJs), which includes decreased vesicle density and organization, decreased quantal release, increased endplate potential duration, and neurofilament (NF) accumulations. This work presents a first study towards defining molecular alterations that may lead to the development of NMJ pathology in SMA. Fast, anterograde transport of synaptic vesicle 2 (SV2-c) and synaptotagmin (Syt1) proteins was reduced 2 days prior to the observed decrease in synaptic vesicle density. Moreover, reduced accumulation of SV2-c or Syt1 was not due to reduced protein expression or reduced kinesin activity. Dynein levels were reduced at times that are consistent with NF accumulations at NMJs. Furthermore, NF distribution, from cell body to sciatic nerve, appeared normal in SMA∆7 mice. Taken together, these results suggest that reduced axonal transport may provide a mechanistic explanation for reduced synaptic vesicle density and concomitant synaptic transmission defects, while providing evidence that suggests NF accumulations result from local NMJ alterations to NFs.

BNP facilitates NMB-encoded histaminergic itch via NPRC-NMBR crosstalk.

Histamine-dependent and -independent itch is conveyed by parallel peripheral neural pathways that express gastrin-releasing peptide (GRP) and neuromedin B (NMB), respectively, to the spinal cord of mice. B-type natriuretic peptide (BNP) has been proposed to transmit both types of itch via its receptor NPRA encoded by Npr1. However, BNP also binds to its cognate receptor, NPRC encoded by Npr3 with equal potency. Moreover, natriuretic peptides (NP) signal through the Gi-couped inhibitory cGMP pathway that is supposed to inhibit neuronal activity, raising the question of how BNP may transmit itch information. Here, we report that Npr3 expression in laminae I-II of the dorsal horn partially overlaps with NMB receptor (NMBR) that transmits histaminergic itch via Gq-couped PLCß-Ca2+ signaling pathway. Functional studies indicate that NPRC is required for itch evoked by histamine but not chloroquine (CQ), a nonhistaminergic pruritogen. Importantly, BNP significantly facilitates scratching behaviors mediated by NMB, but not GRP. Consistently, BNP evoked Ca2+ responses in NMBR/NPRC HEK 293 cells and NMBR/NPRC dorsal horn neurons. These results reveal a previously unknown mechanism by which BNP facilitates NMB-encoded itch through a novel NPRC-NMBR cross-signaling in mice. Our studies uncover distinct modes of action for neuropeptides in transmission and modulation of itch in mice.

An itch is a common sensation that makes us want to scratch. Most short-term itches are caused by histamine, a chemical that is released by immune cells following an infection or in response to an allergic reaction. Chronic itching, on the other hand, is not usually triggered by histamine, and is typically the result of neurological or skin disorders, such as atopic dermatitis. The sensation of itching is generated by signals that travel from the skin to nerve cells in the spinal cord. Studies in mice have shown that the neuropeptides responsible for delivering these signals differ depending on whether or not the itch involves histamine: GRPs (short for gastrin-releasing proteins) convey histamine-independent itches, while NMBs (short for neuromedin B) convey histamine-dependent itches. It has been proposed that another neuropeptide called BNP (short for B-type natriuretic peptide) is able to transmit both types of itch signals to the spinal cord. But it remains unclear how this signaling molecule is able to do this. To investigate, Meng, Liu, Liu, Liu et al. carried out a combination of behavioral, molecular and pharmacological experiments in mice and nerve cells cultured in a laboratory. The experiments showed that BNP alone cannot transmit the sensation of itching, but it can boost itching signals that are triggered by histamine. It is widely believed that BNP activates a receptor protein called NPRA. However, Meng et al. found that the BNP actually binds to another protein which alters the function of the receptor activated by NMBs. These findings suggest that BNP modulates rather than initiates histamine-dependent itching by enhancing the interaction between NMBs and their receptor. Understanding how itch signals travel from the skin to neurons in the spinal cord is crucial for designing new treatments for chronic itching. The work by Meng et al. suggests that treatments targeting NPRA, which was thought to be a key itch receptor, may not be effective against chronic itching, and that other drug targets need to be explored.

Exploration of sensory and spinal neurons expressing gastrin-releasing peptide in itch and pain related behaviors.

Gastrin-releasing peptide (GRP) functions as a neurotransmitter for non-histaminergic itch, but its site of action (sensory neurons vs spinal cord) remains controversial. To determine the role of GRP in sensory neurons, we generated a floxed Grp mouse line. We found that conditional knockout of Grp in sensory neurons results in attenuated non-histaminergic itch, without impairing histamine-induced itch. Using a Grp-Cre knock-in mouse line, we show that the upper epidermis of the skin is exclusively innervated by GRP fibers, whose activation via optogeneics and chemogenetics in the skin evokes itch- but not pain-related scratching or wiping behaviors. In contrast, intersectional genetic ablation of spinal Grp neurons does not affect itch nor pain transmission, demonstrating that spinal Grp neurons are dispensable for itch transmission. These data indicate that GRP is a neuropeptide in sensory neurons for non-histaminergic itch, and GRP sensory neurons are dedicated to itch transmission.

A spinal neural circuitry for converting touch to itch sensation.

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2Cre) receive direct Aß low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2Cre neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.

Pain Inhibits GRPR Neurons via GABAergic Signaling in the Spinal Cord.

It has been known that algogens and cooling could inhibit itch sensation; however, the underlying molecular and neural mechanisms remain poorly understood. Here, we show that the spinal neurons expressing gastrin releasing peptide receptor (GRPR) primarily comprise excitatory interneurons that receive direct and indirect inputs from C and Aδ fibers and form contacts with projection neurons expressing the neurokinin 1 receptor (NK1R). Importantly, we show that noxious or cooling agents inhibit the activity of GRPR neurons via GABAergic signaling. By contrast, capsaicin, which evokes a mix of itch and pain sensations, enhances both excitatory and inhibitory spontaneous synaptic transmission onto GRPR neurons. These data strengthen the role of GRPR neurons as a key circuit for itch transmission and illustrate a spinal mechanism whereby pain inhibits itch by suppressing the function of GRPR neurons.

Spinal Mechanisms of Itch Transmission.

Peripheral itch stimuli are transmitted by sensory neurons to the spinal cord dorsal horn, which then transmits the information to the brain. The molecular and cellular mechanisms within the dorsal horn for itch transmission have only been investigated and identified during the past ten years. This review covers the progress that has been made in identifying the peptide families in sensory neurons and the receptor families in dorsal horn neurons as putative itch transmitters, with a focus on gastrin-releasing peptide (GRP)-GRP receptor signaling. Also discussed are the signaling mechanisms, including opioids, by which various types of itch are transmitted and modulated, as well as the many conflicting results arising from recent studies.

Internode length is reduced during myelination and remyelination by neurofilament medium phosphorylation in motor axons.

The distance between nodes of Ranvier, referred to as internode length, positively correlates with axon diameter, and is optimized during development to ensure maximal neuronal conduction velocity. Following myelin loss, internode length is reestablished through remyelination. However, remyelination results in short internode lengths and reduced conduction rates. We analyzed the potential role of neurofilament phosphorylation in regulating internode length during remyelination and myelination. Following ethidium bromide induced demyelination, levels of neurofilament medium (NF-M) and heavy (NF-H) phosphorylation were unaffected. Preventing NF-M lysine-serine-proline (KSP) repeat phosphorylation increased internode length by 30% after remyelination. To further analyze the role of NF-M phosphorylation in regulating internode length, gene replacement was used to produce mice in which all KSP serine residues were replaced with glutamate to mimic constitutive phosphorylation. Mimicking constitutive KSP phosphorylation reduced internode length by 16% during myelination and motor nerve conduction velocity by ~27% without altering sensory nerve structure or function. Our results suggest that NF-M KSP phosphorylation is part of a cooperative mechanism between axons and Schwann cells that together determine internode length, and suggest motor and sensory axons utilize different mechanisms to establish internode length.

Non-canonical Opioid Signaling Inhibits Itch Transmission in the Spinal Cord of Mice.

Chronic itch or pruritus is a debilitating disorder that is refractory to conventional anti-histamine treatment. Kappa opioid receptor (KOR) agonists have been used to treat chronic itch, but the underlying mechanism remains elusive. Here, we find that KOR and gastrin-releasing peptide receptor (GRPR) overlap in the spinal cord, and KOR activation attenuated GRPR-mediated histamine-independent acute and chronic itch in mice. Notably, canonical KOR-mediated Gαi signaling is not required for desensitizing GRPR function. In vivo and in vitro studies suggest that KOR activation results in the translocation of Ca2+-independent protein kinase C (PKC)δ from the cytosol to the plasma membrane, which in turn phosphorylates and inhibits GRPR activity. A blockade of phospholipase C (PLC) in HEK293 cells prevented KOR-agonist-induced PKCδ translocation and GRPR phosphorylation, suggesting a role of PLC signaling in KOR-mediated GRPR desensitization. These data suggest that a KOR-PLC-PKCδ-GRPR signaling pathway in the spinal cord may underlie KOR-agonists-induced anti-pruritus therapies.

Response to Comment on "Molecular and neural basis of contagious itch behavior in mice".

Liljencrantz et al report the failure of observing contagious itch behavior using mice injected with histamine as the demonstrators. Analysis of their results shows that the histamine model is limited by inadequate frequency and duration of scratching bouts required for contagious itch test. To streamline the contagious itch test, the screen paradigm is highly recommended.

Molecular and neural basis of contagious itch behavior in mice.

Socially contagious itch is ubiquitous in human society, but whether it exists in rodents is unclear. Using a behavioral paradigm that does not entail prior training or reward, we found that mice scratched after observing a conspecific scratching. Molecular mapping showed increased neuronal activity in the suprachiasmatic nucleus (SCN) of the hypothalamus of mice that displayed contagious scratching. Ablation of gastrin-releasing peptide receptor (GRPR) or GRPR neurons in the SCN abolished contagious scratching behavior, which was recapitulated by chemogenetic inhibition of SCN GRP neurons. Activation of SCN GRP/GRPR neurons evoked scratching behavior. These data demonstrate that GRP-GRPR signaling is necessary and sufficient for transmitting contagious itch information in the SCN. The findings may have implications for our understanding of neural circuits that control socially contagious behaviors.

Distinct roles of NMB and GRP in itch transmission.

A key question in our understanding of itch coding mechanisms is whether itch is relayed by dedicated molecular and neuronal pathways. Previous studies suggested that gastrin-releasing peptide (GRP) is an itch-specific neurotransmitter. Neuromedin B (NMB) is a mammalian member of the bombesin family of peptides closely related to GRP, but its role in itch is unclear. Here, we show that itch deficits in mice lacking NMB or GRP are non-redundant and Nmb/Grp double KO (DKO) mice displayed additive deficits. Furthermore, both Nmb/Grp and Nmbr/Grpr DKO mice responded normally to a wide array of noxious stimuli. Ablation of NMBR neurons partially attenuated peripherally induced itch without compromising nociceptive processing. Importantly, electrophysiological studies suggested that GRPR neurons receive glutamatergic input from NMBR neurons. Thus, we propose that NMB and GRP may transmit discrete itch information and NMBR neurons are an integral part of neural circuits for itch in the spinal cord.

Microinjection of histone deacetylase inhibitor into the ventrolateral orbital cortex potentiates morphine induced behavioral sensitization.

Accumulating evidence indicates that epigenetic regulation, such as changes in histone modification in reward-related brain regions, contributes to the memory formation of addiction to opiates and psychostimulants. Our recent results suggested that the ventrolateral orbital cortex (VLO) is involved in the memories of stress and drug addiction. Since addiction and stress memories share some common pathways, the present study was designed to investigate the role of histone deacetylase (HDAC) activity in the VLO during morphine induced-behavioral sensitization. Rats received a single exposure to morphine for establishing the behavioral sensitization model. The effect of HDAC activity in the VLO in morphine induced-behavioral sensitization was examined by microinjection of HDAC inhibitor Trichostatin A (TSA). Furthermore, the protein expression levels of extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK), histone H3 lysine 9 acetylation (aceH3K9) and brain-derived neurotrophic factor (BDNF) in the VLO in morphine-induced behavioral sensitization were examined. The results showed that the bilateral VLO lesions suppressed the expression phase, but not the developmental phase of morphine-induced behavioral sensitization. Microinjection of TSA into the VLO significantly increased both the development and expression phases. Moreover, the protein levels of p-ERK, aceH3K9 and BDNF except ERK in the VLO were significantly upregulated in morphine-treated rats in the expression phase. These effects were further strengthened by intra-VLO injection of TSA. Our findings suggest that HDAC activity in the VLO could potentiate morphine-induced behavioral sensitization. The upregulated expression of p-ERK, aceH3K9 and BDNF in the VLO might be the underlying mechanism of histone acetylation enhancing the morphine-induced behavioral sensitization.