Uptake of gadolinium and dexamethasone in rat inner ear and facial nerve using different administrations.

BACKGROUND: The pathway by which drugs are injected subcutaneously behind the ear to act on the inner ear has not been fully elucidated. OBJECTIVES: To compare the uptake of gadopentetate dimeglumine (Gd-DTPA) and dexamethasone (Dex) in the cochlea and facial nerve of rats following different administrations. MATERIALS AND METHODS: Magnetic resonance imaging was applied to observe the distribution of Gd-DTPA in the facial nerve and inner ear. We observed the uptake of Dex after it was injected with different methods. RESULTS: Images of the intravenous (IV) and intramuscular (IM) groups showed that the bilateral cochlea of the rat was visualized almost simultaneously. While in the left post-auricular (PA) injection group, it was asynchronous. The maximum accumulation (Cmax) of the Gd in the left facial nerve of the PA group (35.406 ± 5.32) was substantially higher than that of the IV group (16.765 ± 3.7542) (p < .01). CONCLUSIONS: Compared with systemic administration, PA has the advantages of long Gd and Dex action time and high accumulation concentration to treat facial nerve diseases. SIGNIFICANCE: The distribution of Gd and Dex in the inner ear and facial nerve of rats following PA injection might be unique.

Hypoxic Bone Mesenchymal Stem Cell-Derived Exosomes Direct Schwann Cells Proliferation, Migration, and Paracrine to Accelerate Facial Nerve Regeneration via circRNA_Nkd2/miR-214-3p/MED19 Axis.

Background: Facial nerves have the potential for regeneration following injury, but this process is often challenging and slow. Schwann cells (SCs) are pivotal in this process. Bone mesenchymal stem cells (BMSC)-derived exosomes promote tissue repair through paracrine action, with hypoxic preconditioning enhancing their effects. The main purpose of this study was to determine whether hypoxia-preconditioned BMSC-derived exosomes (Hypo-Exos) exhibit a greater therapeutic effect on facial nerve repair/regeneration and reveal the mechanism. Methods: CCK-8, EdU, Transwell, and ELISA assays were used to evaluate the functions of Hypo-Exos in SCs. Histological analysis and Vibrissae Movements (VMs) recovery were used to evaluate the therapeutic effects of Hypo-Exos in rat model. circRNA array was used to identify the significantly differentially expressed exosomal circRNAs between normoxia-preconditioned BMSC-derived exosomes (Nor-Exos) and Hypo-Exos. miRDB, TargetScan, double luciferase assay, qRT-PCR and WB were used to predict and identify potential exosomal cirRNA_Nkd2-complementary miRNAs and its target gene. The function of exosomal circRNA_Nkd2 in facial nerve repair/regeneration was evaluated by cell and animal experiments. Results: This study confirmed that Hypo-Exos more effectively promote SCs proliferation, migration, and paracrine function, accelerating facial nerve repair following facial nerve injury (FNI) compared with Nor-Exos. Furthermore, circRNA analysis identified significant enrichment of circRNA_Nkd2 in Hypo-Exos compared with Nor-Exos. Exosomal circRNA_Nkd2 positively regulates mediator complex subunit 19 (MED19) expression by sponging rno-miR-214-3p. Conclusion: Our results demonstrated a mechanism by which Hypo-Exos enhanced SCs proliferation, migration, and paracrine function and facial nerve repair and regeneration following FNI through the circRNA_Nkd2/miR-214-3p/Med19 axis. Hypoxic preconditioning is an effective and promising method for optimizing the therapeutic action of BMSC-derived exosomes in FNI.

SGMS2 in primary osteoporosis with facial nerve palsy.

Pathogenic heterozygous variants in SGMS2 cause a rare monogenic form of osteoporosis known as calvarial doughnut lesions with bone fragility (CDL). The clinical presentations of SGMS2-related bone pathology range from childhood-onset osteoporosis with low bone mineral density and sclerotic doughnut-shaped lesions in the skull to a severe spondylometaphyseal dysplasia with neonatal fractures, long-bone deformities, and short stature. In addition, neurological manifestations occur in some patients. SGMS2 encodes sphingomyelin synthase 2 (SMS2), an enzyme involved in the production of sphingomyelin (SM). This review describes the biochemical structure of SM, SM metabolism, and their molecular actions in skeletal and neural tissue. We postulate how disrupted SM gradient can influence bone formation and how animal models may facilitate a better understanding of SGMS2-related osteoporosis.

Overexpression of c-Jun inhibits erastin-induced ferroptosis in Schwann cells and promotes repair of facial nerve function.

Myelin undergoes various changes after nerve injury, and c-Jun has a close relationship with Schwann cells (SCs). However, it remains unclear whether c-Jun can be involved in nerve repair by regulating ferroptosis. To explore this, we first set up a facial nerve injury model and detected the changes of ferroptosis-related proteins and c-Jun by immunofluorescence and Western blot. Then, we cultured RSC 96 and pSCs, and studied the potential regulatory relationships by a combination of experimental methods such as CCK-8, ELISA, immunofluorescence, qRT-PCR, Western blot and viral transfection. Finally, we corroborated the role of c-Jun through animal experiments. Our experiments revealed that ferroptosis occurs after facial nerve injury. Erastin decreased GPX4, c-Jun proteins and GSH content, while PTGS2, NRF2, HO-1 proteins, MDA, Fe2+ and ROS contents increased. This effect was inhibited after c-Jun overexpression but was reversed after the addition of c-Jun siRNA. Besides, we proved in vivo that c-Jun could inhibit ferroptosis and promote the recovery of facial nerve function. In conclusion, our study identified the relationship between c-Jun and ferroptosis during peripheral nerve injury repair, which provides new ideas for studying peripheral nerve injury and repair.

Primary cutaneous CD8+ cytotoxic T-cell lymphoma of the face with intraoral involvement, resulting in facial nerve palsy after chemotherapy.

The primary cutaneous (PC) CD8+ T-cell lymphoproliferative disorders (LPDs) comprise clinically and histopathologically heterogeneous entities including mycosis fungoides, lymphomatoid papulosis, hydroa-vacciniforme-like LPD, subcutaneous panniculitis-like T-cell lymphoma (TCL), PC acral CD8+ TCL, PC CD8+ aggressive epidermotropic cytotoxic TCL, and PC peripheral TCL, not otherwise specified (PTCL-NOS). We describe a 33-year-old man who presented with progressive facial swelling and lower lip involvement 1 year ago. Microscopy revealed an atypical small to medium-sized lymphoid proliferation exhibiting perivascular accentuation, adnexotropism, and apoptotic cell debris, without surface epithelium involvement. The tumor cells were positive for CD3, CD8, granzyme B, perforin, MUM1/IRF4, and TCR-BF1. The Ki-67 labeling index was 48%. EBER1/2 was negative. Additional studies confirmed localized disease. The diagnosis favored PC-PTCL-NOS. Two months after completing chemotherapy, right-sided facial nerve palsy was diagnosed. CD8+ T-cell LPDs should be considered in the differential diagnosis when assessing facial swelling with intraoral involvement.

DHI Increases the Proliferation and Migration of Schwann Cells Through the PI3K/AKT Pathway and the Expression of CXCL12 and GDNF to Promote Facial Nerve Function Repair.

The facial nerve is one of the vulnerable nerves in otolaryngology. Repair and recovery of facial nerve injury have a high priority in clinical practice. The proliferation and migration of Schwann cells are considered of great significance in the process of nerve injury repair. Danhong injection (DHI), as a common drug for cardiovascular and cerebrovascular diseases, has been fully certified in neuroprotection research, but its role in facial nerve injury is still not clear. Our study found that DHI can promote the proliferation and migration of RSC96 cells, a Schwann cell line, and this effect is related to the activation of the PI3K/AKT pathway. LY294002, an inhibitor of PI3K, inhibits the proliferation and migration of RSC96 cells. Further studies have found that DHI can also promote the expression of CXCL12 and GDNF at gene and protein levels, and CXCL12 is, while GDNF is not, PI3K/AKT pathway-dependent. Animal experiments also confirmed that DHI could promote CXCL12 and GDNF expression and promote facial nerve function recovery and myelin regeneration. In conclusion, our in vitro and in vivo experiments demonstrated that DHI could promote the proliferation and migration of Schwann cells through the PI3K/AKT pathway and increase the expression of CXCL12 and GDNF to promote facial nerve function repair.

Inhibition of nitric oxide production enhances the activity of facial nerve tubulin polymerization and the ability of tau to promote microtubule assembly after neurorrhaphy.

We previously reported that inhibition of nitric oxide (NO) production promotes rat reconnected facial nerve regeneration. However, the underlying mechanism is obscure. Microtubule assembly is known to be essential to axon regeneration; nevertheless, tubulins and microtubule-associated proteins (MAPs) have been demonstrated as targets for NO and peroxynitrite. Thus, we hypothesized that NO and/or peroxynitrite may affect facial nerve regeneration via influencing on microtubule assembly. First, tubulins and tau (a MAP) were extracted from facial nerves of normal rats, treated with NO donor or peroxynitrite, and processed for microtubule assembly assay. We found that peroxynitrite, DEA NONOate, and Angeli's salt reduced the tubulin polymerization activity to a greater extent than GSNO, SIN-1, and SNAP. Additionally, SIN-1, peroxynitrite, and Angeli's salt impaired the ability of tau to promote microtubule assembly. Next, nitrosative stress biomarkers 3-nitrotyrosine (3-NT) and S-nitrosylated cysteine (SNO-Cys) were immunolabeled in facial nerves. Both biomarkers were highly upregulated in proximal and distal stumps of reconnected facial nerves at 3 days and 1 week after neurorrhaphy. Notably, the expression of 3-NT was greatly reduced at 2 weeks, whereas that of SNO-Cys was maintained. Conversely, inhibition of NO production with L-NAME prevented the upregulation of SNO-Cys. Further, we used tubulins and tau extracted from facial nerves of sham-operated, nerve suture + vehicle treatment, and nerve suture + L-NAME treatment rats to perform microtubule assembly assay. We found that L-NAME treatment enhanced polymerization activity of tubulins and ability of tau to promote microtubule assembly. It is noteworthy that α-tubulin plays a more important role than ß-tubulin since the activity of microtubule assembly using α-tubulin extracted from L-NAME-treated rats was greatly elevated, whereas that using ß-tubulin extracted from L-NAME-treated rats was not. Overall, our findings support that inhibition of NO production reduces nitrosative stress, and may thus facilitate microtubule assembly and facial nerve regeneration.

Hedgehog signaling promotes endoneurial fibroblast migration and Vegf-A expression following facial nerve injury.

BACKGROUND: Peripheral nerve injuries are a common clinical problem which may result in permanent loss of motor or sensory function. A better understanding of the signaling pathways that lead to successful nerve regeneration may help in discovering new therapeutic targets. The Hedgehog (Hh) signaling pathway plays significant roles in nerve development and regeneration. In a mouse model of facial nerve injury, Hedgehog-responsive fibroblasts increase in number both at the site of injury and within the distal nerve. However, the role of these cells in facial nerve regeneration is not fully understood. We hypothesize that the Hh pathway plays an angiogenic and pro-migratory role following facial nerve injury. METHODS: Hedgehog pathway modulators were applied to murine endoneurial fibroblasts isolated from the murine facial nerve. The impact of pathway modulation on endoneurial fibroblast migration and cell proliferation was assessed. Gene expression changes of known Hedgehog target genes and the key angiogenic factor Vegf-A were determined by qPCR. In vivo, mice were treated with pathway agonist (SAG21k) and injured facial nerve specimens were analyzed via immunofluorescence and in situ hybridization. RESULTS: Hedgehog pathway activation in facial nerve fibroblasts via SAG21k treatment increases Gli1 and Ptch1 expression, the rate of cellular migration, and Vegf-A expression in vitro. In vivo, expression of Gli1 and Vegf-A expression appears to increase after injury, particularly at the site of nerve injury and the distal nerve, as detected by immunofluorescence and in situ hybridization. Additionally, Gli1 transcripts co-localize with Vegf-A following transection injury to the facial nerve. DISCUSSION: These findings describe an angiogenic and pro-migratory role for the Hedgehog pathway mediated through effects on nerve fibroblasts. Given the critical role of Vegf-A in nerve regeneration, modulation of this pathway may represent a potential therapeutic target to improve facial nerve regeneration following injury.

Prosaposin and its receptors GRP37 and GPR37L1 show increased immunoreactivity in the facial nucleus following facial nerve transection.

Neurotrophic factor prosaposin (PS) is a precursor for saposins A, B, C, and D, which are activators for specific sphingolipid hydrolases in lysosomes. Both saposins and PS are widely contained in various tissues. The brain, skeletal muscle, and heart cells predominantly contain unprocessed PS rather than saposins. PS and PS-derived peptides stimulate neuritogenesis and increase choline acetyltransferase activity in neuroblastoma cells and prevent programmed cell death in neurons. We previously detected increases in PS immunoactivity and its mRNA in the rat facial nucleus following facial nerve transection. PS mRNA expression increased not only in facial motoneurons, but also in microglia during facial nerve regeneration. In the present study, we examined the changes in immunoreactivity of the PS receptors GPR37 and GPR37L1 in the rat facial nucleus following facial nerve transection. Following facial nerve transection, many small Iba1- and glial fibrillary acidic protein (GFAP)-positive cells with strong GPR37L1 immunoreactivity, including microglia and astrocytes, were observed predominately on the operated side. These results indicate that GPR37 mainly works in neurons, whereas GPR37L1 is predominant in microglia or astrocytes, and suggest that increased PS in damaged neurons stimulates microglia or astrocytes via PS receptor GPR37L1 to produce neurotrophic factors for neuronal recovery.

Orexin A and B in the rat superior salivatory nucleus.

Orexin (OX), which regulates sleep and wakefulness and feeding behaviors has 2 isoforms, orexin-A and -B (OXA and OXB). In this study, the distribution of OXA and OXB was examined in the rat superior salivatory nucleus (SSN) using retrograde tracing and immunohistochemical and methods. OXA- and OXB-immunoreactive (-ir) nerve fibers were seen throughout the SSN. These nerve fibers surrounded SSN neurons retrogradely labeled with Fast blue (FB) from the corda-lingual nerve. FB-positive neurons had pericellular OXA- (47.5%) and OXB-ir (49.0%) nerve fibers. Immunohistochemistry for OX receptors also demonstrated the presence of OX1R and OX2R in FB-positive SSN neurons. The majority of FB-positive SSN neurons contained OX1R- (69.7%) or OX2R-immunoreactivity (57.8%). These neurons had small and medium-sized cell bodies. In addition, half of FB-positive SSN neurons which were immunoreactive for OX1R (47.0%) and OX2R (52.2%) had pericellular OXA- and OXB-ir nerve fibers, respectively. Co-expression of OX1R- and OX2R was common in FB-positive SSN neurons. The present study suggests a possibility that OXs regulate the activity of SSN neurons through OX receptors.

CD4+ T cell expression of the IL-10 receptor is necessary for facial motoneuron survival after axotomy.

BACKGROUND: After peripheral nerve transection, facial motoneuron (FMN) survival depends on an intact CD4+ T cell population and a central source of interleukin-10 (IL-10). However, it has not been determined previously whether CD4+ T cells participate in the central neuroprotective IL-10 cascade after facial nerve axotomy (FNA). METHODS: Immunohistochemical labeling of CD4+ T cells, pontine vasculature, and central microglia was used to determine whether CD4+ T cells cross the blood-brain barrier and enter the facial motor nucleus (FMNuc) after FNA. The importance of IL-10 signaling in CD4+ T cells was assessed by performing adoptive transfer of IL-10 receptor beta (IL-10RB)-deficient CD4+ T cells into immunodeficient mice prior to injury. Histology and qPCR were utilized to determine the impact of IL-10RB-deficient T cells on FMN survival and central gene expression after FNA. Flow cytometry was used to determine whether IL-10 signaling in T cells was necessary for their differentiation into neuroprotective subsets. RESULTS: CD4+ T cells were capable of crossing the blood-brain barrier and associating with reactive microglial nodules in the axotomized FMNuc. Full induction of central IL-10R gene expression after FNA was dependent on CD4+ T cells, regardless of their own IL-10R signaling capability. Surprisingly, CD4+ T cells lacking IL-10RB were incapable of mediating neuroprotection after axotomy and promoted increased central expression of genes associated with microglial activation, antigen presentation, T cell co-stimulation, and complement deposition. There was reduced differentiation of IL-10RB-deficient CD4+ T cells into regulatory CD4+ T cells in vitro. CONCLUSIONS: These findings support the interdependence of IL-10- and CD4+ T cell-mediated mechanisms of neuroprotection after axotomy. CD4+ T cells may potentiate central responsiveness to IL-10, while IL-10 signaling within CD4+ T cells is necessary for their ability to rescue axotomized motoneuron survival. We propose that loss of IL-10 signaling in CD4+ T cells promotes non-neuroprotective autoimmunity after FNA.

Nicotinic acetylcholine receptor subunit expression in the gastrocnemius and in the orbicularis oris before and after facial nerve injury in rats.

Objectives: To observe the expression of nicotinic acetylcholine receptor (AChR) subunits in normal orbicularis oris and gastrocnemius muscles and to explore the relationships between the expression of AChR subunits and the severity of facial nerve injury. Methods: Gene and protein expression of AChR subunits in the orbicularis oris and gastrocnemius muscles of male Sprague-Dawley rats was measured by reverse transcription polymerase chain reaction and western blotting, respectively, 1-90 days after graded facial nerve injury. Results: Expression of ε-AChR in the normal orbicularis oris was significantly higher than that in the gastrocnemius, whereas no γ subunit expression was observed. Expression of α, ß, δ, ε, and γ subunits was upregulated in the orbicularis oris and was positively correlated with the degree of facial nerve injury. Discussion: We demonstrated the higher expression of the AChR subunits in the orbicularis oris, compared to gastrocnemius muscles. The differences in expression of these subunits between muscles innervated by the facial nerve and somatic nerves and the correlation of AChR subunit expression with the degree of facial nerve injury yield insights into the sensitivity to muscle relaxants during intraoperative facial nerve monitoring.

Two Pathways Regulate Differential Expression of nAChRs Between the Orbicularis Oris and Gastrocnemius.

BACKGROUND: We previously demonstrated differential expression of nicotinic acetylcholine receptors (nAChRs) in the facial nerve-innervated orbicularis oris and somatic nerve-innervated gastrocnemius, which contribute to different sensitivities to muscle relaxants. Furthermore, the orbicularis oris exhibits less sensitivity to muscle relaxants after facial nerve injury, which is also related to upregulation of nAChRs. Here, we explored the regulatory mechanism for the different expression patterns. Because the agrin/Lrp4/MuSK/rapsyn and neuregulin1/ErbB signaling pathways are indispensable for maintaining the expression of nAChRs, we examined the activity of these two signaling pathways in gastrocnemius and orbicularis oris innervated by normal or injured facial nerves. MATERIALS AND METHODS: A quantitative analysis of these two signaling pathways was realized by immunofluorescence, and immunoprecipitation was applied to detect the level of phosphorylated MuSK in the gastrocnemius and orbicularis oris innervated by normal or injured facial nerves in adult rats. RESULTS: ErbB and the phosphorylated MuSK were expressed more in orbicularis oris than in gastrocnemius (P < 0.05). No significant difference was found in the expression of agrin/Lrp4/MuSK/rapsyn. After facial nerve injury, the level of agrin and the percentage of phosphorylated MuSK decreased significantly, although the expression levels of MuSK, rapsyn, and neuregulin1/ErbB were highly upregulated. CONCLUSIONS: Differential expression of the neuregulin1/ErbB signaling pathway may account for the different expression patterns of nAChRs at the neuromuscular junctions of the orbicularis oris and gastrocnemius. Overexpression of MuSK and rapsyn may contribute to upregulation of nAChRs after facial nerve injury.

CXCL12 has therapeutic value in facial nerve injury and promotes Schwann cells autophagy and migration via PI3K-AKT-mTOR signal pathway.

Facial nerve injury is a clinically common disease accompanied by demyelination of damaged nerves. The remyelination of damaged nerves and the unsatisfactory function recovery are problems that have been plaguing people for a long time. The role that CXCL12 plays after facial nerve injury remains unknown. Our experiments found that the expression of CXCL12 was up-regulated in the early stage of facial nerve injury and decreased after two weeks. Further research found that CXCL12 had no effect on Schwann cells proliferation, apoptosis and cell cycle, while significantly promoted Schwann cells migration. Treatment with CXCL12 decreased the phosphorylation of PI3K, AKT and mTOR, but increased autophagy marker LC3II/I. The CXCL12-induced Schwann cells migration was significantly attenuated by inhibition of autophagy and activation of PI3K pathway through pretreatment with 3-MA and IGF-1 respectively, and this effect was enhanced by PI3K pathway inhibitor LY294002. Animal experiment also confirmed that CXCL12 could improve facial nerve function and myelin regeneration. The findings of this study indicate that CXCL12 can promote the migration of Schwann cells and potentially become a key molecule in the repair of facial nerve injury.

Molecular specification of facial branchial motor neurons in vertebrates.

Orofacial muscles are critical for life-sustaining behaviors, such as feeding and breathing. Centuries of work by neuroanatomists and surgeons resulted in the mapping of bulbar motor neurons in the brainstem and the course of the cranial nerves that carry their axons. Despite the sophisticated understanding of the anatomy of the region, the molecular mechanisms that dictate the development and maturation of facial motor neurons remain poorly understood. This fundamental problem has been recently revisited by physiologists with novel techniques of studying the rhythmic contraction of orofacial muscles in relationship to breathing. The molecular understanding of facial motor neuron development will not only lead to the comprehension of the neural basis of facial expression but may also unlock new avenues to generate stem cell-derived replacements. This review summarizes the current understanding of molecular programs involved in facial motor neuron generation, migration, and maturation, including neural circuit assembly.

Response of the GABAergic System to Axotomy of the Rat Facial Nerve.

The responses of inhibitory neurons/synapses to motoneuron injury in the cranial nervous system remain to be elucidated. In this study, we analyzed GABAA receptor (GABAAR) and GABAergic neurons at the protein level in the transected rat facial nucleus. Immunoblotting revealed that the GABAARα1 protein levels in the axotomized facial nucleus decreased significantly 5-14 days post-insult, and these levels remained low for 5 weeks. Immunohistochemical analysis indicated that the GABAARα1-expressing cells were motoneurons. We next examined the specific components of GABAergic neurons, including glutamate decarboxylase (GAD), vesicular GABA transporter (VGAT) and GABA transporter-1 (GAT-1). Immunoblotting indicated that the protein levels of GAD, VGAT and GAT-1 decreased transiently in the transected facial nucleus from 5 to 14 days post-insult, but returned to the control levels at 5 weeks post-insult. Although GABAARα1 protein levels in the transected nucleus did not return to their control levels for 5 weeks post-insult, the administration of glial cell line-derived neurotrophic factor at the cut site significantly ameliorated the reductions. Through these findings, we verified that the injured facial motoneurons suppressed the levels of GABAARα1 protein over the 5 weeks post-insult, presumably due to the deprivation of neurotrophic factor. On the other hand, the levels of the GAD, VGAT and GAT-1 proteins in GABAergic neurons were transiently reduced in the axotomized facial nucleus at 5-14 days post-insult, but recovered at 4-5 weeks post-insult.

PI3K/Akt and ERK/MAPK Signaling Promote Different Aspects of Neuron Survival and Axonal Regrowth Following Rat Facial Nerve Axotomy.

The ERK/MAPK and PI3K/Akt signaling pathways play important role in neuronal survival and axonal regeneration after peripheral nerve injury. However, the relative importance and degree of functional overlap of the two pathways are still debated due to lack of in-vivo data. We used rats which underwent a facial nerve axotomy, and examined subsequent ERK/MAPK and PI3K/Akt signaling activity by quantifying phosphorylation of ERK and Akt. We also assessed the survival rate of facial neurons, number of regenerated axons, and the length of axonal regrowth in axotomized animals treated with an inhibitor of ERK/MAPK (U0126) or PI3K/Akt (LY294002) phosphorylation, or with vehicle. Axotomy increased phosphorylation of ERK and Akt in the facial nucleus 7 days after injury. The inhibition of ERK phosphorylation significantly reduced the length of regenerated axons, but not the other parameters. Inhibition of Akt phosphorylation significantly reduced the survival rate of facial neurons and the number of new axons, as well as the length of regenerated axons. The results indicate that facial nerve injury activates the ERK/MAPK and PI3K/Akt signaling pathways in the facial nerve nucleus and its axons. However, the pathways promoted aspects of regeneration with only slight overlap: PI3K/Akt signaling improved the survival of neurons, as well as axonal growth and branching, whereas ERK/MAPK signaling promoted only axonal extension.

Progranulin functions as a cathepsin D chaperone to stimulate axonal outgrowth in vivo.

Loss of function mutations in progranulin (GRN) cause frontotemporal dementia, but how GRN haploinsufficiency causes neuronal dysfunction remains unclear. We previously showed that GRN is neurotrophic in vitro. Here, we used an in vivo axonal outgrowth system and observed a delayed recovery in GRN-/- mice after facial nerve injury. This deficit was rescued by reintroduction of human GRN and relied on its C-terminus and on neuronal GRN production. Transcriptome analysis of the facial motor nucleus post injury identified cathepsin D (CTSD) as the most upregulated gene. In aged GRN-/- cortices, CTSD was also upregulated, but the relative CTSD activity was reduced and improved upon exogenous GRN addition. Moreover, GRN and its C-terminal granulin domain granulinE (GrnE) both stimulated the proteolytic activity of CTSD in vitro. Pull-down experiments confirmed a direct interaction between GRN and CTSD. This interaction was also observed with GrnE and stabilized the CTSD enzyme at different temperatures. Investigating the importance of this interaction for axonal regeneration in vivo we found that, although individually tolerated, a combined reduction of GRN and CTSD synergistically reduced axonal outgrowth. Our data links the neurotrophic effect of GRN and GrnE with a lysosomal chaperone function on CTSD to maintain its proteolytic capacity.

Differential regulation of glial reactions in the central facial tract and the facial nucleus after facial neurorrhaphy.

We previously reported that perineuronal astrocytic and microglial reactions are drastically upregulated in the facial nucleus after facial axotomy at the brain stem surface or the stylomastoid foramen. Furthermore, periaxonal astrocytic and microglial reactions develop retrogradely in the central facial tract which contains proximal facial axons in the brain stem. Because reconnection of interrupted peripheral nerve by microsurgical suture is a common clinical practice, the aim of this study was to investigate the spatiotemporal patterns of glial reactions in the central facial tract and the facial nucleus after facial neurorrhaphy. Here, we show immunofluorescent and immunohistochemical evidence that facial neurorrhaphy at the stylomastoid foramen largely prevented axotomy-induced astrocytic and microglial activation in the central facial tract. In contrast, glial reactions in the facial nucleus were still highly elevated after facial neurorrhaphy. Microglial and astrocytic processes were observed to ensheath the facial motoneurons in the facial nucleus. Nevertheless, the transformation of ramified to amoeboid shape of microglia, occurring at 10 weeks after facial axotomy, was not seen after neurorrhaphy. We further examined the effect of N-nitro-l-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), on glial reactions after neurorrhaphy. Western blot analyses demonstrate that inhibition of nitric oxide (NO) production significantly reduced microglial but not astrocytic reaction in the facial nucleus after neurorrhaphy. Taken together, these results indicate that in contrast to the intense glial reactions in both the central facial tract and the facial nucleus after facial axotomy, glial reactions are differentially regulated in these two compartments after facial neurorrhaphy. NO is involved in the activation of microglia in the facial nucleus after facial neurorrhaphy.