A multicenter protocol to assess the prognostic significance of the tumor microenvironment in patients with squamous cell carcinoma of the larynx.

BACKGROUND: The purpose of this multicenter study was to retrospectively investigate the prognostic significance of the tumor microenvironment, in relation to survival in a large cohort of patients with laryngeal squamous cell carcinoma (LSCC), using the method proposed by the International TILs Working Group in breast cancer. METHODS: All consecutive patients with biopsy-proven LSCC who underwent total laryngectomy (TL) between January 2014 and January 2023 were retrospectively included in the study. A retrospective review of medical records including surgical, pathological and follow-up reports was performed. The density of TILs was determined according to the recommendations of the International TILs Working Group. RESULTS: The study group included 186 patients with LSCC. High TILs were statistically correlated with reduced size and extension of primary tumor (pT stage) with a statistically significant value (S: p = 0.01; P: p = 0.0003) and without needs of salvage therapy (S: p = 0.03; P: p = 0.004). Low TILs were indicative of worse prognosis. CONCLUSIONS: Our study confirmed the protective value of TILs and the prognostic role of the tumor microenvironment in LSCC; furthermore, our results showed that the score proposed by the International TILs Working Group for breast cancer can be applied to LSCC.

Tissue expression of miR-449a as risk factor for occult neck metastasis in patients with cT3-T4 N0 laryngeal cancer. A pilot study.

OBJECTIVE: To explore the potential role of miR-449a as biomarker for laryngeal squamous cell carcinoma (LSCC), especially in the decision strategy of neck dissection (ND). METHODS: Each patient underwent total laryngectomy and bilateral ND (levels II-IV); during surgery, tissue samples of around 1 × 0.5 cm were extracted from both healthy tissue adjacent to the tumor and the visibly affected tumor tissue. The extraction of total RNA, encompassing miRNA, was performed using a mirVana PARIS kit. To detect miR449a, cDNA was synthesized from 200 ng of RNA using a TaqMan miRNA reverse transcription kit. RESULTS: The study group was formed of 66 patients (62 males, and 4 females) with LSCC, aged between 39 and 77 years (mean 60 + 14.56 yr). MiR-449a was up-regulated in twenty-eight tumors (42%), while it was down-regulated in 38 samples (58%). In the present study, there was a statistical relevance for miR-449a tissue expression for pN staging (p = 0.017), and PNI (p = 0.005). Eight tumors (12%) cN0 became pN + showing occult cervical lymph node metastases at the final histopathological examination, and all of these patients showed miR-449a downregulation. CONCLUSION: Super-selective ND (sparing the sub evels IIb and IV) might be the approach to cT3-T4 N0 LSCCs with upregulation of miR-449a; on the other hand, to ensure and effective control of occult neck metastases it would be appropriate to reserve elective ND (including sublevels IIb and IV) for cT3-T4 N0 LSCCs with miR-449a downregulation. Although promising, due to the small size of the cohort, the results of this work can be considered preliminary and need to be confirmed by prospective and larger studies.

Adjunctive use of oral MAF is associated with no disease progression or mortality in hospitalized patients with COVID-19 pneumonia: The single-arm COral-MAF1 prospective trial.

Based on a growing body of evidence that a dysregulated innate immune response mediated by monocytes/macrophages plays a key role in the pathogenesis of COVID-19, a clinical trial was conducted to investigate the therapeutic potential and safety of oral macrophage activating factor (MAF) plus standard of care (SoC) in the treatment of hospitalized patients with COVID-19 pneumonia. Ninety-seven hospitalized patients with confirmed COVID-19 pneumonia were treated with oral MAF and a vitamin D3 supplement, in combination with SoC, in a single-arm, open label, multicentre, phase II clinical trial. The primary outcome measure was a reduction in an intensive care unit transfer rate below 13% after MAF administration. At the end of the study, an additional propensity score matching (PSM) analysis was performed to compare the MAF group with a control group treated with SoC alone. Out of 97 patients treated with MAF, none needed care in the ICU and/or intubation with mechanical ventilation or died during hospitalization. Oxygen therapy was discontinued after a median of nine days of MAF treatment. The median length of viral shedding and hospital stay was 14 days and 18 days, respectively. After PSM, statistically significant differences were found in all of the in-hospital outcomes between the two groups. No mild to serious adverse events were recorded during the study. Notwithstanding the limitations of a single-arm study, which prevented definitive conclusions, a 21-day course of MAF treatment plus SoC was found to be safe and promising in the treatment of hospitalized adult patients with COVID-19 pneumonia. Further research will be needed to confirm these preliminary findings.

Prevention of Recurrent Benign Paroxysmal Positional Vertigo: The Role of Combined Supplementation with Vitamin D and Antioxidants.

Benign paroxysmal positional vertigo (BPPV) usually has a favorable course, although it is possible to observe BPPV with a high recurrence rate. Previous studies suggested that vitamin D deficiency might affect BPPV recurrences, and oxidative stress might play a complementary role in BPPV pathogenesis. This multicentric trial aimed to evaluate the effectiveness of oral nutritional supplementation with a compound of alpha-lipoic acid, Carnosine, and Zinc (LICA® (Difass International, Coriano (RN), Italy)), vitamins of group B and vitamin D in preventing BPPV recurrences. A total of 128 patients with high recurrence-BPPV were randomized in three arms: Arm 1 consisted of subjects with "insufficient" or "deficient" vitamin D blood levels, treated with daily oral supplementation of LICA®, vitamins of group B and vitamin D3 (800 UI), Arm 2 included BPPV subjects with "sufficient" vitamin D who did not receive any nutritional support, and Arm 3 included subjects with a "sufficient" serum concentration of vitamin D who received supplementation with a compound of LICA® and Curcumin. After six months of follow-up, a significant reduction of BPPV relapses compared to the baseline was found only in Arm 1 (−2.32, 95% CI: 3.41−1.62, p-value < 0.0001). Study results suggested that oral nutritional supplementation with vitamin D3 plus antioxidants can prevent relapses in patients suffering from high recurrence-BPPV.

Vestibular Disorders after Kidney Transplantation: Focus on the Pathophysiological Mechanisms Underlying the Vertical Nystagmus Associated with Tacrolimus-Related Hypomagnesamia.

The purpose of this paper is to present the case of a patient undergoing kidney transplantation who developed limb tremor dizziness and vertical nystagmus (ny) during Tacrolimus (TAC) therapy and to investigate the pathophysiological mechanisms underlying the balance disorder. This case study regards a 51-year old kidney transplant male patient with hand tremors and lower limbs asthenia associated with dizziness and nausea. The symptoms started two months after the beginning of intravenous TAC for renal transplantation. The pure-tone audiometry showed a mild symmetrical high-frequencies down-sloping sensorineural hearing loss. Acoustic emittance measures showed a normal tympanogram; stapedial reflexes were normally elicited. The Auditory Brainstem Responses (ABR) and Cervical Vestibular Evoked Myogenic Potentials (c-VEMPs) were bilaterally normally evoked. The bedside vestibular examination showed spontaneous down-beating stationary persistent, omni-positional nystagmus, not inhibited by fixation. The Head-Shaking Test accentuates the spontaneous ny. The horizontal clinical head impulse test was negative, bilaterally. A biochemical blood test revealed a decrease in Magnesium (Mg) levels (0.8 mg/dL; normal range 1.58-2.55). The integration of Mg induced both a plasma levels normalization and an improvement of clinical symptoms. This case suggests that TAC treatment can induce a Mg depletion that caused the transient cerebellar lesion. Therefore, the monitoring of serum electrolytes during immunosuppressive treatment appears to be a useful tool in order to reduce the central system symptomatology.

Symptom improvement after transtympanic tube placement in Ménière's disease: preliminary observations.

OBJECTIVE: The treatment of choice for Ménière disease (MD) aims at preventing severity and frequency of vertigo attacks. The purpose of this study was to evaluate the effectiveness of ventilation tube (VT) placement on vertigo control in patients affected by MD with no response to standard medical therapy. METHODS: 76 consecutive outpatients diagnosed with definite MD who failed medical therapy received VT insertion at the Department of Otolaryngology Head and Neck Surgery, "Ospedale del Mare", Naples, Italy, with a 3-year follow up. RESULTS: Over the long term, VT placement was effective in controlling vertigo in 61.8% of patients. In the control group treated with standard preventive care (SPC) alone, all patients continued to experience recurrent vertigo during the entire study. Comparison of survival curves by using the log-rank test shows that significant differences in survival exist between subjects treated with VT placement and the control sample (p = 0.001). CONCLUSIONS: Our long-term follow-up confirms that VT placement is an effective and safe management option in intractable definite MD, especially in the elderly or in those refusing more invasive treatments.

Poorly Differentiated Neuroendocrine Larynx Carcinoma: Clinical Features and miRNAs Signature-A New Goal for Early Diagnosis and Therapy?

Laryngeal neuroendocrine carcinomas (LNECs) are rare and highly heterogeneous malignancies presenting a wide range of pathological and clinical manifestations. Herein, we retrospectively characterize ten patients diagnosticated with LNEC, five of which were defined as well-moderately differentiated neuroendocrine carcinomas, and five that were defined as poorly differentiated neuroendocrine carcinomas, according to the latest WHO classification. Clinical features were analyzed and compared between the two subgroups together with a microRNA study which evidenced a peculiar signature likely related to poorly differentiated larynx neuroendocrine carcinomas. These findings may offer new useful insights for clinicians to improve diagnosis efficiency, therapy response, and patients' outcome for this aggressive neoplasm.

Genetic Manipulation of sn-1-Diacylglycerol Lipase and CB1 Cannabinoid Receptor Gain-of-Function Uncover Neuronal 2-Linoleoyl Glycerol Signaling in Drosophila melanogaster.

Introduction: In mammals, sn-1-diacylglycerol lipases (DAGL) generate 2-arachidonoylglycerol (2-AG) that, as the major endocannabinoid, modulates synaptic neurotransmission by acting on CB1 cannabinoid receptors (CB1R). Even though the insect genome codes for inaE, which is a DAGL ortholog (dDAGL), its products and their functions remain unknown particularly because insects lack chordate-type cannabinoid receptors. Materials and Methods: Gain-of-function and loss-of-function genetic manipulations were carried out in Drosophila melanogaster, including the generation of both dDAGL-deficient and mammalian CB1R-overexpressing flies. Neuroanatomy, dietary manipulations coupled with targeted mass spectrometry determination of arachidonic acid and 2-linoleoyl glycerol (2-LG) production, behavioral assays, and signal transduction profiling for Akt and Erk kinases were employed. Findings from Drosophilae were validated by a CB1R-binding assay for 2-LG in mammalian cortical homogenates with functionality confirmed in neurons using high-throughput real-time imaging in vitro. Results: In this study, we show that dDAGL is primarily expressed in the brain and nerve cord of Drosophila during larval development and in adult with 2-LG being its chief product as defined by dietary precursor availability. Overexpression of the human CB1R in the ventral nerve cord compromised the mobility of adult Drosophilae. The causality of 2-LG signaling to CB1R-induced behavioral impairments was shown by inaE inactivation normalizing defunct motor coordination. The 2-LG-induced activation of transgenic CB1Rs affected both Akt and Erk kinase cascades by paradoxical signaling. Data from Drosophila models were substantiated by showing 2-LG-mediated displacement of [3H]CP 55,940 in mouse cortical homogenates and reduced neurite extension and growth cone collapsing responses in cultured mouse neurons. Conclusions: Overall, these results suggest that 2-LG is an endocannabinoid-like signal lipid produced by dDAGL in Drosophila.

Neck emphysema in a HNSCC cancer patient undergoing concurrent radiotherapy and cetuximab.

BACKGROUND: Lung toxicity in patients undergoing cetuximab and radiotherapy (Cetux-RT) for head and neck squamous cell carcinoma (HNSCC) has been reported in literature and represents a serious side effect of concurrent therapies. METHODS: We report a case of a HNSCC patient that developed neck emphysema during the course of Cetux-RT. The patient was an old male (80 years old) in a good performance status, with an oropharyngeal cancer (T4aN3a). RESULTS: During RT, cone-beam computed tomography (CBCT) showed bilateral neck emphysema that was confirmed at restaging CT. We decided to stop the treatment and to treat the neck emphysema with conservative strategies. After one week CT was repeated and the neck emphysema had improved, so we decided to complete the RT treatment. CONCLUSIONS: Patients undergoing Cetux-RT must be properly selected, whereas IGRT imaging must be viewed carefully in order to permit an early diagnosis and careful management of the patients.

Horizontal semicircular canal jam: Two new cases and possible mechanisms.

INTRODUCTION: Benign paroxysmal positional vertigo (BPPV) of the horizontal semicircular canal (hSCC) can present with otoconia blocking its lumen (canalith jam), with signs and symptoms that make it difficult to distinguish from central nervous system pathology. OBJECTIVE: Here we report two cases of canalith jam affecting the hSCC and offer a theoretical mechanism based on known vestibular neurophysiology. METHODS: We use video-oculography to document the canalith jam and show the moment the otoconia loosen. RESULTS: Canalith jam is a rare form of BPPV remedied with repositioning maneuvers. CONCLUSION: Clinicians should consider canalith jam as a mechanism for BPPV when the nystagmus is (a) Direction fixed with fixation removed and during positional testing; (b) Velocity dependent on supine head position; (c) Converts to geotropic directional changing nystagmus.

A TRPV1-to-secretagogin regulatory axis controls pancreatic ß-cell survival by modulating protein turnover.

Ca2+-sensor proteins are generally implicated in insulin release through SNARE interactions. Here, secretagogin, whose expression in human pancreatic islets correlates with their insulin content and the incidence of type 2 diabetes, is shown to orchestrate an unexpectedly distinct mechanism. Single-cell RNA-seq reveals retained expression of the TRP family members in ß-cells from diabetic donors. Amongst these, pharmacological probing identifies Ca2+-permeable transient receptor potential vanilloid type 1 channels (TRPV1) as potent inducers of secretagogin expression through recruitment of Sp1 transcription factors. Accordingly, agonist stimulation of TRPV1s fails to rescue insulin release from pancreatic islets of glucose intolerant secretagogin knock-out(-/-) mice. However, instead of merely impinging on the SNARE machinery, reduced insulin availability in secretagogin-/- mice is due to ß-cell loss, which is underpinned by the collapse of protein folding and deregulation of secretagogin-dependent USP9X deubiquitinase activity. Therefore, and considering the desensitization of TRPV1s in diabetic pancreata, a TRPV1-to-secretagogin regulatory axis seems critical to maintain the structural integrity and signal competence of ß-cells.

Secretagogin-dependent matrix metalloprotease-2 release from neurons regulates neuroblast migration.

The rostral migratory stream (RMS) is viewed as a glia-enriched conduit of forward-migrating neuroblasts in which chemorepulsive signals control the pace of forward migration. Here we demonstrate the existence of a scaffold of neurons that receive synaptic inputs within the rat, mouse, and human fetal RMS equivalents. These neurons express secretagogin, a Ca2+-sensor protein, to execute an annexin V-dependent externalization of matrix metalloprotease-2 (MMP-2) for reconfiguring the extracellular matrix locally. Mouse genetics combined with pharmacological probing in vivo and in vitro demonstrate that MMP-2 externalization occurs on demand and that its loss slows neuroblast migration. Loss of function is particularly remarkable upon injury to the olfactory bulb. Cumulatively, we identify a signaling cascade that provokes structural remodeling of the RMS through recruitment of MMP-2 by a previously unrecognized neuronal constituent. Given the life-long presence of secretagogin-containing neurons in human, this mechanism might be exploited for therapeutic benefit in rescue strategies.

Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signalling.

Local environmental cues are indispensable for axonal growth and guidance during brain circuit formation. Here, we combine genetic and pharmacological tools, as well as systems neuroanatomy in human fetuses and mouse models, to study the role of endocannabinoid and Slit/Robo signalling in axonal growth. We show that excess 2-arachidonoylglycerol, an endocannabinoid affecting directional axonal growth, triggers corpus callosum enlargement due to the errant CB1 cannabinoid receptor-containing corticofugal axon spreading. This phenotype mechanistically relies on the premature differentiation and end-feet proliferation of CB2R-expressing oligodendrocytes. We further show the dependence of both axonal Robo1 positioning and oligodendroglial Slit2 production on cell-type-specific cannabinoid receptor activation. Accordingly, Robo1 and/or Slit2 manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can configure focal Slit2/Robo1 signalling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic deficits and prenatal drug exposure.

Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury.

Neuronal calcium (Ca(2+))-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca(2+)-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca(2+)-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca(2+)-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.

Miswiring the brain: Δ9-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway.

Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ(9)-tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor-driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC-sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin-2, a microtubule-binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis-exposed fetuses, defining SCG10 as the first cannabis-driven molecular effector in the developing cerebrum. CB1 cannabinoid receptor activation recruits c-Jun N-terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring.

Targeted lipidomics in Drosophila melanogaster identifies novel 2-monoacylglycerols and N-acyl amides.

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.

Nerve growth factor scales endocannabinoid signaling by regulating monoacylglycerol lipase turnover in developing cholinergic neurons.

Endocannabinoid, particularly 2-arachidonoyl glycerol (2-AG), signaling has recently emerged as a molecular determinant of neuronal migration and synapse formation during cortical development. However, the cell type specificity and molecular regulation of spatially and temporally confined morphogenic 2-AG signals remain unexplored. Here, we demonstrate that genetic and pharmacological manipulation of CB(1) cannabinoid receptors permanently alters cholinergic projection neuron identity and hippocampal innervation. We show that nerve growth factor (NGF), implicated in the morphogenesis and survival of cholinergic projection neurons, dose-dependently and coordinately regulates the molecular machinery for 2-AG signaling via tropomyosine kinase A receptors in vitro. In doing so, NGF limits the sorting of monoacylglycerol lipase (MGL), rate limiting 2-AG bioavailability, to proximal neurites, allowing cell-autonomous 2-AG signaling at CB(1) cannabinoid receptors to persist at atypical locations to induce superfluous neurite extension. We find that NGF controls MGL degradation in vitro and in vivo and identify the E3 ubiquitin ligase activity of breast cancer type 1 susceptibility protein (BRCA1) as a candidate facilitating MGL's elimination from motile neurite segments, including growth cones. BRCA1 inactivation by cisplatin or genetically can rescue and reposition MGL, arresting NGF-induced growth responses. These data indicate that NGF can orchestrate endocannabinoid signaling to promote cholinergic differentiation and implicate BRCA1 in determining neuronal morphology.

Dietary energy substrates reverse early neuronal hyperactivity in a mouse model of Alzheimer's disease.

Deficient energy metabolism and network hyperactivity are the early symptoms of Alzheimer's disease (AD). In this study, we show that administration of exogenous oxidative energy substrates (OES) corrects neuronal energy supply deficiency that reduces the amyloid-beta-induced abnormal neuronal activity in vitro and the epileptic phenotype in AD model in vivo. In vitro, acute application of protofibrillar amyloid-ß1-42 (Aß1-42) induced aberrant network activity in wild-type hippocampal slices that was underlain by depolarization of both the neuronal resting membrane potential and GABA-mediated current reversal potential. Aß1-42 also impaired synaptic function and long-term potentiation. These changes were paralleled by clear indications of impaired energy metabolism, as indicated by abnormal NAD(P)H signaling induced by network activity. However, when glucose was supplemented with OES pyruvate and 3-beta-hydroxybutyrate, Aß1-42 failed to induce detrimental changes in any of the above parameters. We administered the same OES as chronic supplementation to a standard diet to APPswe/PS1dE9 transgenic mice displaying AD-related epilepsy phenotype. In the ex-vivo slices, we found neuronal subpopulations with significantly depolarized resting and GABA-mediated current reversal potentials, mirroring abnormalities we observed under acute Aß1-42 application. Ex-vivo cortex of transgenic mice fed with standard diet displayed signs of impaired energy metabolism, such as abnormal NAD(P)H signaling and strongly reduced tolerance to hypoglycemia. Transgenic mice also possessed brain glycogen levels twofold lower than those of wild-type mice. However, none of the above neuronal and metabolic dysfunctions were observed in transgenic mice fed with the OES-enriched diet. In vivo, dietary OES supplementation abated neuronal hyperexcitability, as the frequency of both epileptiform discharges and spikes was strongly decreased in the APPswe/PS1dE9 mice placed on the diet. Altogether, our results suggest that early AD-related neuronal malfunctions underlying hyperexcitability and energy metabolism deficiency can be prevented by dietary supplementation with native energy substrates.

Secretagogin is expressed in sensory CGRP neurons and in spinal cord of mouse and complements other calcium-binding proteins, with a note on rat and human.

BACKGROUND: Secretagogin (Scgn), a member of the EF-hand calcium-binding protein (CaBP) superfamily, has recently been found in subsets of developing and adult neurons. Here, we have analyzed the expression of Scgn in dorsal root ganglia (DRGs) and trigeminal ganglia (TGs), and in spinal cord of mouse at the mRNA and protein levels, and in comparison to the well-known CaBPs, calbindin D-28k, parvalbumin and calretinin. Rat DRGs, TGs and spinal cord, as well as human DRGs and spinal cord were used to reveal phylogenetic variations. RESULTS: We found Scgn mRNA expressed in mouse and human DRGs and in mouse ventral spinal cord. Our immunohistochemical data showed a complementary distribution of Scgn and the three CaBPs in mouse DRG neurons and spinal cord. Scgn was expressed in ~7% of all mouse DRG neuron profiles, mainly small ones and almost exclusively co-localized with calcitonin gene-related peptide (CGRP). This co-localization was also seen in human, but not in rat DRGs. Scgn could be detected in the mouse sciatic nerve and accumulated proximal to its constriction. In mouse spinal cord, Scgn-positive neuronal cell bodies and fibers were found in gray matter, especially in the dorsal horn, with particularly high concentrations of fibers in the superficial laminae, as well as in cell bodies in inner lamina II and in some other laminae. A dense Scgn-positive fiber network and some small cell bodies were also found in the superficial dorsal horn of humans. In the ventral horn, a small number of neurons were Scgn-positive in mouse but not rat, confirming mRNA distribution. Both in mouse and rat, a subset of TG neurons contained Scgn. Dorsal rhizotomy strongly reduced Scgn fiber staining in the dorsal horn. Peripheral axotomy did not clearly affect Scgn expression in DRGs, dorsal horn or ventral horn neurons in mouse. CONCLUSIONS: Scgn is a CaBP expressed in a subpopulation of nociceptive DRG neurons and their processes in the dorsal horn of mouse, human and rat, the former two co-expressing CGRP, as well as in dorsal horn neurons in all three species. Functional implications of these findings include the cellular refinement of sensory information, in particular during the processing of pain.

Differential subcellular recruitment of monoacylglycerol lipase generates spatial specificity of 2-arachidonoyl glycerol signaling during axonal pathfinding.

Endocannabinoids, particularly 2-arachidonoyl glycerol (2-AG), impact the directional turning and motility of a developing axon by activating CB(1) cannabinoid receptors (CB(1)Rs) in its growth cone. Recent findings posit that sn-1-diacylglycerol lipases (DAGLα/ß) synthesize 2-AG in the motile axon segment of developing pyramidal cells. Coincident axonal targeting of CB(1)Rs and DAGLs prompts the hypothesis that autocrine 2-AG signaling facilitates axonal outgrowth. However, DAGLs alone are insufficient to account for the spatial specificity and dynamics of 2-AG signaling. Therefore, we hypothesized that local 2-AG degradation by monoacylglycerol lipase (MGL) must play a role. We determined how subcellular recruitment of MGL is temporally and spatially restricted to establish the signaling competence of 2-AG during axonal growth. MGL is expressed in central and peripheral axons of the fetal nervous system by embryonic day 12.5. MGL coexists with DAGLα and CB(1)Rs in corticofugal axons of pyramidal cells. Here, MGL and DAGLα undergo differential axonal targeting with MGL being excluded from the motile neurite tip. Thus, spatially confined MGL activity generates a 2-AG-sensing microdomain and configures 2-AG signaling to promote axonal growth. Once synaptogenesis commences, MGL disperses in stationary growth cones. The axonal polarity of MGL is maintained by differential proteasomal degradation because inhibiting the ubiquitin proteasome system also induces axonal MGL redistribution. Because MGL inactivation drives a CB(1)R-dependent axonal growth response, we conclude that 2-AG may act as a focal protrusive signal for developing neurons and whose regulated metabolism is critical for attaining correct axonal complexity.