A novel macrolide-Del-1 axis to regenerate bone in old age.

Aging is associated with increased susceptibility to chronic inflammatory bone loss disorders, such as periodontitis, in large part due to the impaired regenerative potential of aging tissues. DEL-1 exerts osteogenic activity and promotes bone regeneration. However, DEL-1 expression declines with age. Here we show that systemically administered macrolide antibiotics and a non-antibiotic erythromycin derivative, EM-523, restore DEL-1 expression in 18-month-old ("aged") mice while promoting regeneration of bone lost due to naturally occurring age-related periodontitis. These compounds failed to induce bone regeneration in age-matched DEL-1-deficient mice. Consequently, these drugs promoted DEL-1-dependent functions, including alkaline phosphatase activity and osteogenic gene expression in the periodontal tissue while inhibiting osteoclastogenesis, leading to net bone growth. Macrolide-treated aged mice exhibited increased skeletal bone mass, suggesting that this treatment may be pertinent to systemic bone loss disorders. In conclusion, we identified a macrolide-DEL-1 axis that can regenerate bone lost due to aging-related disease.

A SARM1-mitochondrial feedback loop drives neuropathogenesis in a Charcot-Marie-Tooth disease type 2A rat model.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an axonal neuropathy caused by mutations in the mitofusin 2 (MFN2) gene. MFN2 mutations result in profound mitochondrial abnormalities, but the mechanism underlying the axonal pathology is unknown. Sterile α and Toll/IL-1 receptor motif-containing 1 (SARM1), the central executioner of axon degeneration, can induce neuropathy and is activated by dysfunctional mitochondria. We tested the role of SARM1 in a rat model carrying a dominant CMT2A mutation (Mfn2H361Y) that exhibits progressive dying-back axonal degeneration, neuromuscular junction (NMJ) abnormalities, muscle atrophy, and mitochondrial abnormalities - all hallmarks of the human disease. We generated Sarm1-KO (Sarm1-/-) and Mfn2H361Y Sarm1 double-mutant rats and found that deletion of Sarm1 rescued axonal, synaptic, muscle, and functional phenotypes, demonstrating that SARM1 was responsible for much of the neuropathology in this model. Despite the presence of mutant MFN2 protein in these double-mutant rats, loss of SARM1 also dramatically suppressed many mitochondrial defects, including the number, size, and cristae density defects of synaptic mitochondria. This surprising finding indicates that dysfunctional mitochondria activated SARM1 and that activated SARM1 fed back on mitochondria to exacerbate the mitochondrial pathology. As such, this work identifies SARM1 inhibition as a therapeutic candidate for the treatment of CMT2A and other neurodegenerative diseases with prominent mitochondrial pathology.

Macrophage depletion blocks congenital SARM1-dependent neuropathy.

Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified 2 rare NMNAT2 missense variants in 2 brothers afflicted with a progressive neuropathy syndrome. The polymorphisms resulted in amino acid substitutions V98M and R232Q, which reduced NMNAT2 NAD+-synthetase activity. We generated a mouse model to mirror the human syndrome and found that Nmnat2V98M/R232Q compound-heterozygous CRISPR mice survived to adulthood but developed progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes were all SARM1-dependent. Remarkably, macrophage depletion therapy blocked and reversed neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induced inflammatory neuropathy and highlight the potential of immune therapy as a treatment for this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

Loss of Stathmin-2, a hallmark of TDP-43-associated ALS, causes motor neuropathy.

TDP-43 mediates proper Stathmin-2 (STMN2) mRNA splicing, and STMN2 protein is reduced in the spinal cord of most patients with amyotrophic lateral sclerosis (ALS). To test the hypothesis that STMN2 loss contributes to ALS pathogenesis, we generated constitutive and conditional STMN2 knockout mice. Constitutive STMN2 loss results in early-onset sensory and motor neuropathy featuring impaired motor behavior and dramatic distal neuromuscular junction (NMJ) denervation of fast-fatigable motor units, which are selectively vulnerable in ALS, without axon or motoneuron degeneration. Selective excision of STMN2 in motoneurons leads to similar NMJ pathology. STMN2 knockout heterozygous mice, which better model the partial loss of STMN2 protein found in patients with ALS, display a slowly progressive, motor-selective neuropathy with functional deficits and NMJ denervation. Thus, our findings strongly support the hypothesis that STMN2 reduction owing to TDP-43 pathology contributes to ALS pathogenesis.

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss.

SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might activate SARM1. Here, we show the nicotinamide analog 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN, which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration, and neuronal death. In mice, systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to the peripheral nerve induces SARM1-dependent axon degeneration. We identify 2-aminopyridine as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity and suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

Perivascular Hedgehog responsive cells play a critical role in peripheral nerve regeneration via controlling angiogenesis.

Hh signaling has been shown to be activated in intact and injured peripheral nerve. However, the role of Hh signaling in peripheral nerve is not fully understood. In the present study, we observed that Hh signaling responsive cells [Gli1(+) cells] in both the perineurium and endoneurium. In the endoneurium, Gli1(+) cells were classified as blood vessel associated or non-associated. After injury, Gli1(+) cells around blood vessels mainly proliferated to then accumulate into the injury site along with endothelial cells. Hh signaling activity was retained in Gli1(+) cells during nerve regeneration. To understand the role of Hedgehog signaling in Gli1(+) cells during nerve regeneration, we examined mice with Gli1(+) cells-specific inactivation of Hh signaling (Smo cKO). After injury, Smo cKO mice showed significantly reduced numbers of accumulated Gli1(+) cells along with disorganized vascularization at an early stage of nerve regeneration, which subsequently led to an abnormal extension of the axon. Thus, Hh signaling in Gli1(+) cells appears to be involved in nerve regeneration through controlling new blood vessel formation at an early stage.

Rapid elevation of brain-derived neurotrophic factor production in the bilateral trigeminal ganglia by unilateral transection of the mental nerve in mice.

OBJECTIVES: Previous spinal nerve injury studies have reported brain-derived neurotrophic factor (BDNF) mRNA upregulation in either the ipsilateral dorsal root ganglion (DRG) neurons or both the contralateral and ipsilateral DRG neurons from early period after peripheral nerve injury. This BDNF elevation induces hyperalgesia in the injured and/or uninjured sites, but this detailed mechanism remains unknown. This study aimed to investigate the BDNF mRNA expression in bilateral DRG neurons caused by unilateral nerve injury and to explore the possible mechanisms by which nitric oxide (NO) mediates BDNF production in the DRG, resulting in contralateral hyperalgesia. METHODS: Early changes in BDNF mRNA expression in the bilateral trigeminal ganglia, within 1 day after mental nerve transection, were examined. Additionally, the effects on BDNF production of the NO synthase inhibitor N(ω)-nitro-l-arginine methyl ester (L-NAME) were investigated in the bilateral trigeminal ganglia. The relationship between injured neurons and BDNF production in the trigeminal ganglia was then assessed using immunohistochemical and retrograde tracing methods. RESULTS: Reverse transcription-PCR analysis demonstrated that unilateral transection of the mental nerve induced a rapid elevation of BDNF mRNA expression, which was inhibited by the intracerebroventricular administration of L-NAME prior to nerve transection. This effect was observed in both the ipsilateral and contralateral sides to the nerve transection. BDNF immunostaining combined with FluoroGold retrograde tracing revealed two types of BDNF-reactive neurons, FluoroGold-labelled and non-FluoroGold-labelled neurons, in the ipsilateral and contralateral sides of the trigeminal ganglia. BDNF-positive cells were also observed in the trigeminal ganglia of other trigeminal nerve branches. CONCLUSIONS: Unilateral nerve injury upregulates BDNF production in the bilateral trigeminal ganglia by NO-mediated and/or indirect activation of afferent neurons, resulting in contralateral hyperalgesia.

Overactivation of the NF-κB pathway impairs molar enamel formation.

OBJECTIVE: Hypohidrotic ectodermal dysplasia (HED) is a hereditary disorder characterized by abnormal structures and functions of the ectoderm-derived organs, including teeth. HED patients exhibit a variety of dental symptoms, such as hypodontia. Although disruption of the EDA/EDAR/EDARADD/NF-κB pathway is known to be responsible for HED, it remains unclear whether this pathway is involved in the process of enamel formation. EXPERIMENTAL SUBJECTS AND METHODS: To address this question, we examined the mice overexpressing Ikkß (an essential component required for the activation of NF-κB pathway) under the keratin 5 promoter (K5-Ikkß). RESULTS: Upregulation of the NF-κB pathway was confirmed in the ameloblasts of K5-Ikkß mice. Premature abrasion was observed in the molars of K5-Ikkß mice, which was accompanied by less mineralized enamel. However, no significant changes were observed in the enamel thickness and the pattern of enamel rods in K5-Ikkß mice. Klk4 expression was significantly upregulated in the ameloblasts of K5-Ikkß mice at the maturation stage, and the expression of its substrate, amelogenin, was remarkably reduced. This suggests that abnormal enamel observed in K5-Ikkß mice was likely due to the compromised degradation of enamel protein at the maturation stage. CONCLUSION: Therefore, we could conclude that the overactivation of the NF-κB pathway impairs the process of amelogenesis.

Gli3 is a Key Factor in the Schwann Cells from Both Intact and Injured Peripheral Nerves.

Hedgehog (Hh) signaling has been shown to be involved in regulating both intact and injured peripheral nerves. Therefore, it is critical to understand how Hh signaling is regulated in the peripheral nerve. One of the transcription factors of the Hh signaling pathway, Gli3, functions as both a repressor and an activator of Hh signaling activity. However, it remains unclear whether Gli3 is involved in controlling the intact and/or injured peripheral nerves. We found that Gli3 act as a repressor in the Schwann cells (SCs) of intact sciatic nerves. Although Dhh and Ptch1 expression were present, Hh signaling was not activated in these SCs. Moreover, heterozygous Gli3 mutation (Gli3-/+) induced ectopic Hh signaling activity in SCs. Hh signaling was thus suppressed by Gli3 in the SCs of intact sciatic nerves. Minor morphological changes were observed in the intact nerves from Gli3-/+ mice. Gli3 expression was significantly decreased following injury and ligand expression switched from Dhh to Shh, which activated Hh signaling in SCs from wild-type mice. Changes of these ligands was found to be important for nerve regeneration in which the downregulation of Gli3 was also involved. In fact, Gli3-/+ mice exhibited accelerated ligand switching and subsequent nerve regeneration. Both suppression of Hh signaling with Gli3 in the intact nerves and activation of Hh signaling without Gli3 in the injured nerve were observed in the SCs in an autocrine manner. Thus, Gli3 is a key factor in the control of intact peripheral nerve homeostasis and nerve regeneration.

Endotracheal Intubation Complicated by a Palatal Tooth in a Patient With Treacher Collins Syndrome.

We report a case of difficult endotracheal intubation in a patient with Treacher Collins syndrome. A sixteen-year-old female patient scheduled for general anesthesia had a displaced palatal tooth that interfered with laryngoscope insertion into the pharyngeal space. To address this problem, we successfully performed endotracheal intubation using a fiberscope while elevating the epiglottic vallecula using a King Vision™ video laryngoscope. A later operation was performed after tooth extraction without difficult laryngoscopy. Our experience stresses the importance of removing obstructions to laryngoscopic inspection prior to general anesthesia.

Semaphorin 3A Inhibits Nerve Regeneration During Early Stage after Inferior Alveolar Nerve Transection.

Neuroma formation at sites of injury can impair peripheral nerve regeneration. Although the involvement of semaphorin 3A has been suggested in neuroma formation, this detailed process after injury is not fully understood. This study was therefore undertaken to examine the effects of semaphorin 3A on peripheral nerve regeneration during the early stage after injury. Immunohistochemistry for semaphorin 3A and PGP9.5, a general neuronal marker, was carried out for clarify chronological changes in their expressions after transection of the mouse inferior alveolar nerve thorough postoperative days 1 to 7. At postoperative day 1, the proximal stump of the damaged IAN exhibited semaphorin 3A, while the distal stump lacked any immunoreactivity. From this day on, its expression lessened, ultimately disappearing completely in all regions of the transected inferior alveolar nerve. A local administration of an antibody to semaphorin 3A into the nerve transection site at postoperative day 3 inhibited axon sprouting at the injury site. This antibody injection increased the number of trigeminal ganglion neurons labeled with DiI (paired t-test, p < 0.05). Immunoreactivity of the semaphorin 3A receptor, neuropilin-1, was also detected at the proximal stump at postoperative day 1. These results suggest that nerve injury initiates semaphorin 3A production in ganglion neurons, which is then delivered through the nerve fibers to the proximal end, thereby contributes to the inhibition of axonal sprouting from the proximal region of injured nerves in the distal direction. To our knowledge, this is the first report to reveal the involvement of Sema3A in the nerve regeneration process at its early stage.

MicroRNAs control eyelid development through regulating Wnt signaling.

BACKGROUND: The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. RESULTS: It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing; Dicer fl/fl ;Wnt1Cre) leads to the EOB phenotype with full penetrance. Here, we identified that the up-regulation of Wnt signaling resulted in the EOB phenotype in Dicer mutants. Down-regulation of Fgf signaling observed in Dicer mutants was caused by an inverse relationship between Fgf and Wnt signaling. Shh and Bmp signaling were down-regulated as the secondary effects in Dicer fl/fl ;Wnt1Cre mice. Wnt, Shh, and Fgf signaling were also found to mediate the epithelial-mesenchymal interactions in eyelid development. CONCLUSIONS: miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.

Vascularization via activation of VEGF-VEGFR signaling is essential for peripheral nerve regeneration.

Peripheral nerve injury has been suggested to up-regulate mRNA for the vascular endothelial growth factor (VEGF) which enhances nerve regeneration. VEGF is known to regulate angiogenesis by binding with a specific receptor, the vascular endothelial growth factor receptor (VEGFR). However, little is known about the involvement of VEGF-VEGFR signaling in the nerve regeneration at early stages though previous studies contained a lengthy observation. The present study examined that relationship between angiogenesis and peripheral nerve regeneration at the early stage after nerve transection by focusing on the chronological changes in the expression patterns of VEGF-VEGFR signaling. This study used our previously reported experimental model for nerve regeneration following the transection of the inferior alveolar nerve (IAN) in mice. In a double staining of PGP9.5 and CD31, respective markers for the nerve fibers and endothelial cells, CD31 immunoreactions first appeared in the injury site on postoperative (PO) day 2 when the transected nerve fibers had not been re-connected. The most intense immunoreaction for CD31 was found around the regenerating nerve fibers extending from the proximal stump on PO day 3, but it gradually lessened to disappear by PO day 7. The expression patterns of VEGFR1 and VEGFR2 showed similar chronological changes through the observation periods, with most intense immunoreaction found on PO day 3. Western blotting of total protein extracted from the injury site demonstrated the clear bands for VEGF-A and VEGF-B on PO day 2, indicating a time lag for the expression of ligands and receptors. A local administration of antibody to VEGF-A inhibited the elongation of the nerve fibers from the proximal stump. Furthermore, this administration of VEGF-A antibody inhibited the expression of CD31 in the gap between proximal and distal stumps. These results indicated that a nerve injury initiates productions in VEGF-A and VEFG-B, followed with the expression of VEGFR1 and VEGFR2 at early stages after the nerve injury. Taken these findings together, it is reasonable to postulate that immediate response of VEGF-VEGFR signaling to nerve injury plays a crucial role in local angiogenesis, resulting in a trigger for the regeneration of the nerve fibers in mouse IAN.

The Sonic Hedgehog signaling pathway regulates inferior alveolar nerve regeneration.

Activation of Shh signaling is known to be observed following injury of the peripheral nerves such as the sciatic nerve. However, the precise role of Shh signaling during peripheral nerve regeneration is not fully understood. The inferior alveolar nerve (IAN) is most commonly injured during oral surgery. Unlike the sciatic nerve, the IAN is isolated from other craniofacial tissues, as it resides in a long bony canal within the mandible. The IAN is thus an excellent experimental model for investigating peripheral nerve regeneration. In this study, the role of Shh signaling in peripheral nerve regeneration was investigated using the mouse IAN transection model. During regeneration, Shh signaling was activated within the entire distal region of the IAN and proximal stumps. Inhibition of Shh signaling by cyclopamine application at the transection site led to abnormal axon growth in random directions, a reduced number of macrophages, and an increase in myelin debris within the distal region. Shh signaling is thus involved in peripheral nerve regeneration via the regulation of myelin degradation.

Locally Produced BDNF Promotes Sclerotic Change in Alveolar Bone after Nerve Injury.

Brain-derived neurotrophic factor (BDNF), which is released due to nerve injury, is known to promote the natural healing of injured nerves. It is often observed that damage of mandibular canal induces local sclerotic changes in alveolar bone. We reported that peripheral nerve injury promotes the local production of BDNF; therefore, it was possible to hypothesize that peripheral nerve injury affects sclerotic changes in the alveolar bone. This study aimed to evaluate the effect of BDNF on osteogenesis using in vitro osteoblast-lineage cell culture and an in vivo rat osteotomy model. MC3T3-E1 cells were cultured with BDNF and were examined for cell proliferative activity, chemotaxis and mRNA expression levels of osteoblast differentiation markers. For in vivo study, inferior alveolar nerve (IAN) injury experiments and mandibular cortical osteotomy were performed using a rat model. In the osteotomy model, exogenous BDNF was applied to bone surfaces after corticotomy of the mandible, and we morphologically analyzed the new bone formation. As a result, mRNA expression of osteoblast differentiation marker, osteocalcin, was significantly increased by BDNF, although cell proliferation and migration were not affected. In the in vivo study, osteopontin-positive new bone formation was significantly accelerated in the BDNF-grafted groups, and active bone remodeling, involving trkB-positive osteoblasts and osteocytes, continued after 28 days. In conclusion, BDNF stimulated the differentiation of MC3T3-E1 cells and it promoted new bone formation and maturation. These results suggested that local BDNF produced by peripheral nerve injury contributes to accelerating sclerotic changes in the alveolar bone.

Peripheral Brain-Derived Neurotrophic Factor Modulates Regeneration Following Inferior Alveolar Nerve Injury in Rats.

AIMS: To examine the effects of local brain-derived neurotrophic factor (BDNF) produced after nerve injury on the functional regeneration of the damaged nerve. METHODS: The inferior alveolar nerve was transected in adult male rats and 1 µg or 10 µg of BDNF antibody was administered at the injury site; a third group of rats received saline and a fourth group underwent nerve ligation. BDNF mRNA was quantified in the transected tissue and trigeminal ganglion by using real-time polymerase chain reaction (PCR). Head withdrawal thresholds following mechanical (tactile) stimulation (with von Frey filaments) of the mental region were measured for 3 weeks postoperatively. Electromyographic activity of the jaw opening reflex (JOR) was recorded from the anterior belly of the digastric muscle. RESULTS: Within 24 hours, transection induced significant elevation of BDNF mRNA expression in the injured tissue (unpaired t test, P < .01). The head withdrawal threshold to mechanical stimulation increased at 1 day after transection and then decreased (two-way repeated measures analysis of variance [ANOVA], P < .001). At 2 weeks after surgery, the head withdrawal threshold was higher than before surgery in the group that received a higher dose of BDNF antibody (ANOVA, P < .001), but not in the group that received a smaller dose (ANOVA, P > .05). No significant differences were observed in the latency or threshold of the JOR between saline- and antibody-treated rats (unpaired t test, P > .05). CONCLUSION: These results suggest that locally administered BDNF antibody neutralizes nerve injury-induced BDNF at the injury site and thus influences sensorimotor recovery.