Natural Killer Cells Degenerate Intact Sensory Afferents following Nerve Injury.

Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D, Retinoic Acid Early 1 (RAE1), is re-expressed in adult dorsal root ganglion neurons following peripheral nerve injury, triggering selective degeneration of injured axons. Infiltration of cytotoxic NK cells into the sciatic nerve by extravasation occurs within 3 days following crush injury. Using a combination of genetic cell ablation and cytokine-antibody complex stimulation, we show that NK cell function correlates with loss of sensation due to degeneration of injured afferents and reduced incidence of post-injury hypersensitivity. This neuro-immune mechanism of selective NK cell-mediated degeneration of damaged but intact sensory axons complements Wallerian degeneration and suggests the therapeutic potential of modulating NK cell function to resolve painful neuropathy through the clearance of partially damaged nerves.

TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation.

Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(ßγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(ßγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 µM, whereas slow mode targets neuronal NMDA receptors at around 1 µM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.

Splice-dependent trans-synaptic PTPδ-IL1RAPL1 interaction regulates synapse formation and non-REM sleep.

Alternative splicing regulates trans-synaptic adhesions and synapse development, but supporting in vivo evidence is limited. PTPδ, a receptor tyrosine phosphatase adhering to multiple synaptic adhesion molecules, is associated with various neuropsychiatric disorders; however, its in vivo functions remain unclear. Here, we show that PTPδ is mainly present at excitatory presynaptic sites by endogenous PTPδ tagging. Global PTPδ deletion in mice leads to input-specific decreases in excitatory synapse development and strength. This involves tyrosine dephosphorylation and synaptic loss of IL1RAPL1, a postsynaptic partner of PTPδ requiring the PTPδ-meA splice insert for binding. Importantly, PTPδ-mutant mice lacking the PTPδ-meA insert, and thus lacking the PTPδ interaction with IL1RAPL1 but not other postsynaptic partners, recapitulate biochemical and synaptic phenotypes of global PTPδ-mutant mice. Behaviorally, both global and meA-specific PTPδ-mutant mice display abnormal sleep behavior and non-REM rhythms. Therefore, alternative splicing in PTPδ regulates excitatory synapse development and sleep by modulating a specific trans-synaptic adhesion.

Ibuprofen-induced multiorgan malformation during embryogenesis in Xenopus laevis (FETAX).

Ibuprofen, one of the most commonly prescribed nonsteroidal anti-inflammatory drugs, has not been fully assessed for embryonic toxicity in vertebrates. Here, we systematically assessed the embryotoxicity of ibuprofen in Xenopus laevis at various concentrations during embryogenesis. Embryos were treated with different concentrations of ibuprofen, ranging from 8 to 64 mg/L, at 23 °C for 96 h, and examined daily and evaluated at 72 hpf. Lethal or teratogenic effects were documented. For histological analysis, paraffin embedded embryos were transversely sectioned at a thickness of 10-µm and stained with hematoxylin and eosin. Total RNA was isolated from embryos at stages 6, 12, 22 and 36, and real-time quantitative PCR was performed. Ibuprofen-treated embryos showed delayed or failed dorsal lip formation and its closure at the beginning of gastrulation. This resulted in herniation of the endodermal mass after gastrulation under high concentrations of ibuprofen-treated embryos. Underdeveloped intestines with stage and/or intestinal malrotation, distorted microcephaly, and hypoplastic heart, lungs, and pronephric tubules were observed in ibuprofen-treated embryos. Cephalic, cardiac, and truncal edema were also observed in them. The severity of the deformities was observed in a concentration-dependent manner. The teratogenic index was 2.28. These gross and histological disruptions correlated well with the altered expression of each organ marker gene. In conclusion, ibuprofen induced delayed and disrupted gastrulation in the early developmental stage and multiorgan malformation later in the organogenesis stage of Xenopus laevis embryos.

Cerebellar Nuclei Receiving Orofacial Proprioceptive Signals through the Mossy Fiber Pathway from the Supratrigeminal Nucleus in Rats.

Proprioception from muscle spindles is necessary for motor function executed by the cerebellum. In particular, cerebellar nuclear neurons that receive proprioceptive signals and send projections to the lower brainstem or spinal cord play key roles in motor control. However, little is known about which cerebellar nuclear regions receive orofacial proprioception. Here, we investigated projections to the cerebellar nuclei from the supratrigeminal nucleus (Su5), which conveys the orofacial proprioception arising from jaw-closing muscle spindles (JCMSs). Injections of an anterograde tracer into the Su5 resulted in a large number of labeled axon terminals bilaterally in the dorsolateral hump (IntDL) of the cerebellar interposed nucleus (Int) and the dorsolateral protuberance (MedDL) of the cerebellar medial nucleus. In addition, a moderate number of axon terminals were ipsilaterally labeled in the vestibular group Y nucleus (group Y). We electrophysiologically detected JCMS proprioceptive signals in the IntDL and MedDL. Retrograde tracing analysis confirmed bilateral projections from the Su5 to the IntDL and MedDL. Furthermore, anterograde tracer injections into the external cuneate nucleus (ECu), which receives other proprioceptive input from forelimb/neck muscles, resulted in only a limited number of ipsilaterally labeled terminals, mainly in the dorsomedial crest of the Int and the group Y. Taken together, the Su5 and ECu axons almost separately terminated in the cerebellar nuclei (except for partial overlap in the group Y). These data suggest that orofacial proprioception is differently processed in the cerebellar circuits in comparison to other body-part proprioception, thus contributing to the executive function of orofacial motor control.

The Cerebellar Cortex Receives Orofacial Proprioceptive Signals from the Supratrigeminal Nucleus via the Mossy Fiber Pathway in Rats.

Proprioceptive sensory information from muscle spindles is essential for the regulation of motor functions. However, little is known about the motor control regions in the cerebellar cortex that receive proprioceptive signals from muscle spindles distributed throughout the body, including the orofacial muscles. Therefore, in this study, we investigated the pattern of projections in the rat cerebellar cortex derived from the supratrigeminal nucleus (Su5), which conveys orofacial proprioceptive information from jaw-closing muscle spindles (JCMSs). Injections of an anterograde tracer into the Su5 revealed that many bilateral axon terminals (rosettes) were distributed in the granular layer of the cerebellar cortex (including the simple lobule B, crus II and flocculus) in a various sized, multiple patchy pattern. We could also detect JCMS proprioceptive signals in these cerebellar cortical regions, revealing for the first time that they receive muscle proprioceptive inputs in rats. Retrograde tracer injections confirmed that the Su5 directly sends outputs to the cerebellar cortical areas. Furthermore, we injected an anterograde tracer into the external cuneate nucleus (ECu), which receives proprioceptive signals from the forelimb and neck muscle spindles, to distinguish between the Su5- and ECu-derived projections in the cerebellar cortex. The labeled terminals from the ECu were distributed predominantly in the vermis of the cerebellar cortex. Almost no overlap was seen in the terminal distributions of the Su5 and ECu projections. Our findings demonstrate that the rat cerebellar cortex receives orofacial proprioceptive input that is processed differently from the proprioceptive signals from the other regions of the body.

Early correction of synaptic long-term depression improves abnormal anxiety-like behavior in adult GluN2B-C456Y-mutant mice.

Extensive evidence links Glutamate receptor, ionotropic, NMDA2B (GRIN2B), encoding the GluN2B/NR2B subunit of N-methyl-D-aspartate receptors (NMDARs), with various neurodevelopmental disorders, including autism spectrum disorders (ASDs), but the underlying mechanisms remain unclear. In addition, it remains unknown whether mutations in GluN2B, which starts to be expressed early in development, induces early pathophysiology that can be corrected by early treatments for long-lasting effects. We generated and characterized Grin2b-mutant mice that carry a heterozygous, ASD-risk C456Y mutation (Grin2b+/C456Y). In Grin2b+/C456Y mice, GluN2B protein levels were strongly reduced in association with decreased hippocampal NMDAR currents and NMDAR-dependent long-term depression (LTD) but unaltered long-term potentiation, indicative of mutation-induced protein degradation and LTD sensitivity. Behaviorally, Grin2b+/C456Y mice showed normal social interaction but exhibited abnormal anxiolytic-like behavior. Importantly, early, but not late, treatment of young Grin2b+/C456Y mice with the NMDAR agonist D-cycloserine rescued NMDAR currents and LTD in juvenile mice and improved anxiolytic-like behavior in adult mice. Therefore, GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, NMDAR-dependent LTD, and anxiety-like behavior, and early activation of NMDAR function has long-lasting effects on adult mouse behavior.

Protective Effect of Ciclopirox against Ovariectomy-Induced Bone Loss in Mice by Suppressing Osteoclast Formation and Function.

Postmenopausal osteoporosis is closely associated with excessive osteoclast formation and function, resulting in the loss of bone mass. Osteoclast-targeting agents have been developed to manage this disease. We examined the effects of ciclopirox on osteoclast differentiation and bone resorption in vitro and in vivo. Ciclopirox significantly inhibited osteoclast formation from primary murine bone marrow macrophages (BMMs) in response to receptor activator of nuclear factor kappa B ligand (RANKL), and the expression of genes associated with osteoclastogenesis and function was decreased. The formation of actin rings and resorption pits was suppressed by ciclopirox. Analysis of RANKL-mediated early signaling events in BMMs revealed that ciclopirox attenuates IκBα phosphorylation without affecting mitogen-activated protein kinase activation. Furthermore, the administration of ciclopirox suppressed osteoclast formation and bone loss in ovariectomy-induced osteoporosis in mice and reduced serum levels of osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus. These results indicate that ciclopirox exhibits antiosteoclastogenic activity both in vitro and in vivo and represents a new candidate compound for protection against osteoporosis and other osteoclast-related bone diseases.

PF-3845, a Fatty Acid Amide Hydrolase Inhibitor, Directly Suppresses Osteoclastogenesis through ERK and NF-κB Pathways In Vitro and Alveolar Bone Loss In Vivo.

Alveolar bone loss, the major feature of periodontitis, results from the activation of osteoclasts, which can consequently cause teeth to become loose and fall out; the development of drugs capable of suppressing excessive osteoclast differentiation and function is beneficial for periodontal disease patients. Given the difficulties associated with drug discovery, drug repurposing is an efficient approach for identifying alternative uses of commercially available compounds. Here, we examined the effects of PF-3845, a selective fatty acid amide hydrolase (FAAH) inhibitor, on receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclastogenesis, its function, and the therapeutic potential for the treatment of alveolar bone destruction in experimental periodontitis. PF-3845 significantly suppressed osteoclast differentiation and decreased the induction of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and the expression of osteoclast-specific markers. Actin ring formation and osteoclastic bone resorption were also reduced by PF-3845, and the anti-osteoclastogenic and anti-resorptive activities were mediated by the suppression of phosphorylation of rapidly accelerated fibrosarcoma (RAF), mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase, (ERK) and nuclear factor κB (NF-κB) inhibitor (IκBα). Furthermore, the administration of PF-3845 decreased the number of osteoclasts and the amount of alveolar bone destruction caused by ligature placement in experimental periodontitis in vivo. The present study provides evidence that PF-3845 is able to suppress osteoclastogenesis and prevent alveolar bone loss, and may give new insights into its role as a treatment for osteoclast-related diseases.

Lrfn2-Mutant Mice Display Suppressed Synaptic Plasticity and Inhibitory Synapse Development and Abnormal Social Communication and Startle Response.

SALM1 (SALM (synaptic adhesion-like molecule), also known as LRFN2 (leucine rich repeat and fibronectin type III domain containing), is a postsynaptic density (PSD)-95-interacting synaptic adhesion molecule implicated in the regulation of NMDA receptor (NMDAR) clustering largely based on in vitro data, although its in vivo functions remain unclear. Here, we found that mice lacking SALM1/LRFN2 (Lrfn2-/- mice) show a normal density of excitatory synapses but altered excitatory synaptic function, including enhanced NMDAR-dependent synaptic transmission but suppressed NMDAR-dependent synaptic plasticity in the hippocampal CA1 region. Unexpectedly, SALM1 expression was detected in both glutamatergic and GABAergic neurons and Lrfn2-/- CA1 pyramidal neurons showed decreases in the density of inhibitory synapses and the frequency of spontaneous inhibitory synaptic transmission. Behaviorally, ultrasonic vocalization was suppressed in Lrfn2-/- pups separated from their mothers and acoustic startle was enhanced, but locomotion, anxiety-like behavior, social interaction, repetitive behaviors, and learning and memory were largely normal in adult male Lrfn2-/- mice. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, and social communication and startle behaviors in mice.SIGNIFICANCE STATEMENT Synaptic adhesion molecules regulate synapse development and function, which govern neural circuit and brain functions. The SALM/LRFN (synaptic adhesion-like molecule/leucine rich repeat and fibronectin type III domain containing) family of synaptic adhesion proteins consists of five known members for which the in vivo functions are largely unknown. Here, we characterized mice lacking SALM1/LRFN2 (SALM1 KO) known to associate with NMDA receptors (NMDARs) and found that these mice showed altered NMDAR-dependent synaptic transmission and plasticity, as expected, but unexpectedly also exhibited suppressed inhibitory synapse development and synaptic transmission. Behaviorally, SALM1 KO pups showed suppressed ultrasonic vocalization upon separation from their mothers and SALM1 KO adults showed enhanced responses to loud acoustic stimuli. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, social communication, and acoustic startle behavior.

5-HT2A receptor activation enhances NMDA receptor-mediated glutamate responses through Src kinase in the dendrites of rat jaw-closing motoneurons.

KEY POINTS: 5-HT increases the excitability of brainstem and spinal motoneurons, including the jaw-closing motoneurons, by depolarizing the membrane potential and decreasing the medium-duration afterhyperpolarization. In this study, we focused on how 5-HT enhances postsynaptic glutamatergic responses in the dendrites of the jaw-closing motoneurons. We demonstrate that 5-HT augments glutamatergic signalling by enhancing the function of the GluN2A-containing NMDA receptor (NMDAR) through the activation of 5-HT2A receptors (5-HT2A Rs) and Src kinase. To enhance glutamatergic responses, activation of the 5-HT2A Rs must occur within ∼60 µm of the location of the glutamate responses. 5-HT inputs to the jaw-closing motoneurons can significantly vary their input-output relationship, which may contribute to wide-range regulation of contractile forces of the jaw-closing muscles. ABSTRACT: Various motor behaviours are modulated by 5-HT. Although the masseter (jaw-closing) motoneurons receive both glutamatergic and serotonergic inputs, it remains unclear how 5-HT affects the glutamatergic inputs to the motoneuronal dendrites. We examined the effects of 5-HT on postsynaptic responses evoked by single- or two-photon uncaging of caged glutamate (glutamate responses) to the dendrites of masseter motoneurons in postnatal day 2-5 rats of either sex. Application of 5-HT induced membrane depolarization and enhanced the glutamate-response amplitude. This enhancement was mimicked by the 5-HT2A receptor (5-HT2A R) agonist and was blocked by the 5-HT2A/2C R antagonist. However, neither the 5-HT2B R nor the 5-HT2C R agonists altered glutamate responses. Blockade of the NMDA receptors (NMDARs), but not AMPA receptors, abolished the 5-HT-induced enhancement. Furthermore, the selective antagonist for the GluN2A subunit abolished the 5-HT-induced enhancement. 5-HT increased GluN2A phosphorylation, while the Src kinase inhibitor reduced the 5-HT-induced enhancement and GluN2A phosphorylation. When exposure to the 5-HT2A R agonist was targeted to the dendrites, the enhancement of glutamate responses was restricted to the loci of the dendrites near the puff loci. Electron microscopic immunohistochemistry revealed that both the NMDARs and the 5-HT2A Rs were close to each other in the same dendrite. These results suggest that activation of dendritic 5-HT2A Rs enhances the function of local GluN2A-containing NMDARs through Src kinase. Such enhancement of the glutamate responses by 5-HT may contribute to wide-range regulation of contractile forces of the jaw-closing muscles.

Increasing ERK phosphorylation by inhibition of p38 activity protects against cadmium-induced apoptotic cell death through ERK/Drp1/p38 signaling axis in spermatocyte-derived GC-2spd cells.

Many studies report that cadmium chloride (CdCl2)-induces oxidative stress is associated with male reproductive damage in the testes. CdCl2 also induces mitochondrial fission by increasing dynamin-related protein 1 (Drp1) expression as well as the mitochondria-dependent apoptosis pathway by extracellular signal-regulated kinase (ERK) activation. However, it remains unclear whether mechanisms linked to the mitochondrial damage signal via CdCl2-induced mitogen-activated protein kinases (MAPK) cause damage to spermatocytes. In this study, increased intracellular and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (∆Ψm) depolarization, and mitochondrial fragmentation and swelling were observed at 5 µM of CdCl2 exposure, resulting in increased apoptotic cell death. Moreover, CdCl2-induced cell death is closely associated with the ERK/Drp1/p38 signaling axis. Interestingly, SB203580, a p38 inhibitor, effectively prevented CdCl2-induced apoptotic cell death by reducing ∆Ψm depolarization and intracellular and mitochondrial ROS levels. Knockdown of Drp1 expression diminished CdCl2-induced mitochondrial deformation and ROS generation and protected GC-2spd cells from apoptotic cell death. In addition, electron microscopy showed that p38 inhibition reduced CdCl2-induced mitochondrial interior damage more effectively than N-acetyl-L-cysteine (NAC), an ROS scavenger; ERK inhibition; or Drp1 knockdown. Therefore, these results demonstrate that inhibition of p38 activity prevents CdCl2-induced apoptotic GC-2spd cell death by reducing depolarization of mitochondrial membrane potential and mitochondrial ROS levels via ERK phosphorylation in a signal pathway different from the CdCl2-induced ERK/Drp1/p38 axis and suggest a therapeutic strategy for CdCl2-induced male infertility.

Peroxiredoxin 4 ameliorates amyloid beta oligomer-mediated apoptosis by inhibiting ER-stress in HT-22 hippocampal neuron cells.

Alzheimer's disease (AD) is a neurodegenerative disorder caused by amyloid beta oligomers (AßO), which induce cell death by triggering oxidative stress and endoplasmic reticulum (ER) stress. Oxidative stress is regulated by antioxidant enzymes, including peroxiredoxins. Peroxiredoxins (Prx) are classified into six subtypes, based on their localization and cysteine residues, and protect cells by scavenging hydrogen peroxide (H2O2). Peroxiredoxin 4 (Prx4) is unique in being localized to the ER; however, whether Prx4 protects neuronal cells from AßO-induced toxicity remains unclear, although Prx4 expression is upregulated in AßO-induced oxidative stress and ER stress. In this study, we established HT-22 cells in which Prx4 was either overexpressed or silenced to investigate its role in AßO-induced toxicity. AßO-stimulation of HT-22 cells with overexpressed Prx4 caused decreases in both AßO-induced ROS and ER stress (followed by ER expansion). In contrast, AßO stimulation caused increases in both ROS and ER stress that were notably higher in HT-22 cells with silenced Prx4 expression than in HT-22 cells. Consequently, Prx4 overexpression decreased apoptotic cell death and ameliorated the AßO-induced increase in intracellular Ca2+. Therefore, we conclude that Prx4 has a protective effect against AßO-mediated oxidative stress, ER stress, and neuronal cell death. Furthermore, these results suggest that Prx4 may be a target for preventing AßO toxicity in AD. Graphical abstract .

Evaluation of the toxic effects of celecoxib on Xenopus embryo development.

Celecoxib is a non-steroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2 and is prescribed for severe pain and inflammation. The excellent therapeutic effects of celecoxib mean that it is frequently used clinically, including for women of child-bearing age. However, the prenatal effects of this compound have not been studied extensively in vertebrates. The present study examined the developmental toxicity of celecoxib using a frog embryo teratogenic assay-Xenopus (FETAX). In addition, we examined its effects on cell migration using co-cultures of human umbilical vein endothelial cells and 10T1/2 cells. These studies revealed that celecoxib induced concentration-dependent mortality and various malformations of the Xenopus internal organs, including gut miscoiling, haemorrhage, and oedema. Celecoxib also downregulated the expression of vascular wall markers (Msr and alpha smooth muscle actin) and other organ-specific markers (Nkx2.5, Cyl104 and IFABP). In vitro co-culture studies revealed that celecoxib inhibited pericyte migration and differentiation into vascular smooth muscle cells. In conclusion, celecoxib was both toxic and teratogenic in Xenopus embryos, where it produced serious heart and vessel malformation by inhibiting vascular wall maturation and vascular network formation.

Inhibitory effects of methyl-3,5-di-O-caffeoyl-epi-quinate on RANKL-induced osteoclast differentiation.

In this study, we have shown that methyl-3,5-di-O-caffeoyl-epi-quinate, a naturally occurring compound isolated from Ainsliaea acerifolia, inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and the expression of osteoclast marker genes. Methyl-3,5-di-O-caffeoyl-epi-quinate also inhibited RANKL-induced activation of p38, Akt and extracellular signal-regulated kinase (ERK) as well as the expression of nuclear factor of activated T-cell (NFATc1), the key regulator of osteoclast differentiation. Negative regulators for osteoclast differentiation was upregulated by methyl-3,5-di-O-caffeoyl-epi-quinate. Collectively, our results suggested that methyl-3,5-di-O-caffeoyl-epi-quinate suppresses osteoclast differentiation via downregulation of RANK signaling pathways and NFATc1.

Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function.

Autism spectrum disorder (ASD) is a group of conditions characterized by impaired social interaction and communication, and restricted and repetitive behaviours. ASD is a highly heritable disorder involving various genetic determinants. Shank2 (also known as ProSAP1) is a multi-domain scaffolding protein and signalling adaptor enriched at excitatory neuronal synapses, and mutations in the human SHANK2 gene have recently been associated with ASD and intellectual disability. Although ASD-associated genes are being increasingly identified and studied using various approaches, including mouse genetics, further efforts are required to delineate important causal mechanisms with the potential for therapeutic application. Here we show that Shank2-mutant (Shank2(-/-)) mice carrying a mutation identical to the ASD-associated microdeletion in the human SHANK2 gene exhibit ASD-like behaviours including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping. These mice show a marked decrease in NMDA (N-methyl-D-aspartate) glutamate receptor (NMDAR) function. Direct stimulation of NMDARs with D-cycloserine, a partial agonist of NMDARs, normalizes NMDAR function and improves social interaction in Shank2(-/-) mice. Furthermore, treatment of Shank2(-/-) mice with a positive allosteric modulator of metabotropic glutamate receptor 5 (mGluR5), which enhances NMDAR function via mGluR5 activation, also normalizes NMDAR function and markedly enhances social interaction. These results suggest that reduced NMDAR function may contribute to the development of ASD-like phenotypes in Shank2(-/-) mice, and mGluR modulation of NMDARs offers a potential strategy to treat ASD.

Preemptive application of QX-314 attenuates trigeminal neuropathic mechanical allodynia in rats.

The aim of the present study was to examine the effects of preemptive analgesia on the development of trigeminal neuropathic pain. For this purpose, mechanical allodynia was evaluated in male Sprague-Dawley rats using chronic constriction injury of the infraorbital nerve (CCI-ION) and perineural application of 2% QX-314 to the infraorbital nerve. CCI-ION produced severe mechanical allodynia, which was maintained until postoperative day (POD) 30. An immediate single application of 2% QX-314 to the infraorbital nerve following CCI-ION significantly reduced neuropathic mechanical allodynia. Immediate double application of QX-314 produced a greater attenuation of mechanical allodynia than a single application of QX-314. Immediate double application of 2% QX-314 reduced the CCI-ION-induced upregulation of GFAP and p-p38 expression in the trigeminal ganglion. The upregulated p-p38 expression was co-localized with NeuN, a neuronal cell marker. We also investigated the role of voltage-gated sodium channels (Navs) in the antinociception produced by preemptive application of QX-314 through analysis of the changes in Nav expression in the trigeminal ganglion following CCI-ION. Preemptive application of QX-314 significantly reduced the upregulation of Nav1.3, 1.7, and 1.9 produced by CCI-ION. These results suggest that long-lasting blockade of the transmission of pain signaling inhibits the development of neuropathic pain through the regulation of Nav isoform expression in the trigeminal ganglion. Importantly, these results provide a potential preemptive therapeutic strategy for the treatment of neuropathic pain after nerve injury.

Cerebellar Shank2 Regulates Excitatory Synapse Density, Motor Coordination, and Specific Repetitive and Anxiety-Like Behaviors.

Shank2 is a multidomain scaffolding protein implicated in the structural and functional coordination of multiprotein complexes at excitatory postsynaptic sites as well as in psychiatric disorders, including autism spectrum disorders. While Shank2 is strongly expressed in the cerebellum, whether Shank2 regulates cerebellar excitatory synapses, or contributes to the behavioral abnormalities observed in Shank2-/- mice, remains unexplored. Here we show that Shank2-/- mice show reduced excitatory synapse density in cerebellar Purkinje cells in association with reduced levels of excitatory postsynaptic proteins, including GluD2 and PSD-93, and impaired motor coordination in the Erasmus test. Shank2 deletion restricted to Purkinje cells (Pcp2-Cre;Shank2fl/fl mice) leads to similar reductions in excitatory synapse density, synaptic protein levels, and motor coordination. Pcp2-Cre;Shank2fl/fl mice do not recapitulate autistic-like behaviors observed in Shank2-/- mice, such as social interaction deficits, altered ultrasonic vocalizations, repetitive behaviors, and hyperactivity. However, Pcp2-Cre;Shank2fl/fl mice display enhanced repetitive behavior in the hole-board test and anxiety-like behavior in the light-dark test, which are not observed in Shank2-/- mice. These results implicate Shank2 in the regulation of cerebellar excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors. SIGNIFICANCE STATEMENT: The postsynaptic side of excitatory synapses contains multiprotein complexes, termed the postsynaptic density, which contains receptors, scaffolding/adaptor proteins, and signaling molecules. Shank2 is an excitatory postsynaptic scaffolding protein implicated in the formation and functional coordination of the postsynaptic density and has been linked to autism spectrum disorders. Using Shank2-null mice and Shank2-conditional knock-out mice with a gene deletion restricted to cerebellar Purkinje cells, we explored functions of Shank2 in the cerebellum. We found that Shank2 regulates excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors, but is not associated with autistic-like social deficits or repetitive behaviors.

Participation of central GABAA receptors in the trigeminal processing of mechanical allodynia in rats.

Here we investigated the central processing mechanisms of mechanical allodynia and found a direct excitatory link with low-threshold input to nociceptive neurons. Experiments were performed on male Sprague-Dawley rats weighing 230-280 g. Subcutaneous injection of interleukin 1 beta (IL-1ß) (1 ng/10 µL) was used to produce mechanical allodynia and thermal hyperalgesia. Intracisternal administration of bicuculline, a gamma aminobutyric acid A (GABAA) receptor antagonist, produced mechanical allodynia in the orofacial area under normal conditions. However, intracisternal administration of bicuculline (50 ng) produced a paradoxical anti-allodynic effect under inflammatory pain conditions. Pretreatment with resiniferatoxin (RTX), which depletes capsaicin receptor protein in primary afferent fibers, did not alter the paradoxical anti-allodynic effects produced by the intracisternal injection of bicuculline. Intracisternal injection of bumetanide, an Na-K-Cl cotransporter (NKCC 1) inhibitor, reversed the IL-1ß-induced mechanical allodynia. In the control group, application of GABA (100 µM) or muscimol (3 µM) led to membrane hyperpolarization in gramicidin perforated current clamp mode. However, in some neurons, application of GABA or muscimol led to membrane depolarization in the IL-1ß-treated rats. These results suggest that some large myelinated Aß fibers gain access to the nociceptive system and elicit pain sensation via GABAA receptors under inflammatory pain conditions.

Methionine sulfoxide reductase B3 deficiency causes hearing loss due to stereocilia degeneration and apoptotic cell death in cochlear hair cells.

Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically reduces methionine-R-sulfoxide to methionine. A recent genetic study showed that the MSRB3 gene is associated with autosomal recessive hearing loss in human deafness DFNB74. However, the precise role of MSRB3 in the auditory system and the pathogenesis of hearing loss have not yet been determined. This work is the first to generate MsrB3 knockout mice to elucidate the possible pathological mechanisms of hearing loss observed in DFNB74 patients. We found that homozygous MsrB3(-/-) mice were profoundly deaf and had largely unaffected vestibular function, whereas heterozygous MsrB3(+/-) mice exhibited normal hearing similar to that of wild-type mice. The MsrB3 protein is expressed in the sensory epithelia of the cochlear and vestibular tissues, beginning at E15.5 and E13.5, respectively. Interestingly, MsrB3 is densely localized at the base of stereocilia on the apical surface of auditory hair cells. MsrB3 deficiency led to progressive degeneration of stereociliary bundles starting at P8, followed by a loss of hair cells, resulting in profound deafness in MsrB3(-/-) mice. The hair cell loss appeared to be mediated by apoptotic cell death, which was measured using TUNEL and caspase 3 immunocytochemistry. Taken together, our data suggest that MsrB3 plays an essential role in maintaining the integrity of hair cells, possibly explaining the pathogenesis of DFNB74 deafness in humans caused by MSRB3 deficiency.