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Background: Neuroligin-3 is a postsynaptic adhesion molecule involved in synapse development and function. It is implicated in rare, monogenic forms of autism, and its shedding is critical to the tumor microenvironment of gliomas. While other members of the neuroligin family exhibit synapse-type specificity in localization and function through distinct interactions with postsynaptic scaffold proteins, the specificity of neuroligin-3 synaptic localization remains largely unknown., Methods: We investigated the synaptic localization of neuroligin-3 across regions in mouse and human brain samples after validating antibody specificity in knockout animals. We raised a phospho-specific neuroligin antibody and used phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types., Results: Neuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. We identified specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses., Conclusions: These data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses. These findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition., (Copyright © 2023 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Microrobots are of significant interest due to their smart transport capabilities, especially for precisely targeted delivery in dynamic environments (blood, cell membranes, tumor interstitial matrixes, blood-brain barrier, mucosa, and other body fluids). To perform a more complex micromanipulation in biological applications, it is highly desirable for microrobots to be stimulated with multiple stimuli rather than a single stimulus. Herein, the biodegradable and biocompatible smart micromotors with a Janus architecture consisting of PrecirolATO 5 and polycaprolactone compartments inspired by the anisotropic geometry of tadpoles and sperms are newly designed. These bioinspired micromotors combine the advantageous properties of polypyrrole nanoparticles (NPs), a high near-infrared light-absorbing agent with high photothermal conversion efficiency, and magnetic NPs, which respond to the magnetic field and exhibit multistimulus-responsive behavior. By combining both fields, we achieved an "on/off" propulsion mechanism that can enable us to overcome complex tasks and limitations in liquid environments and overcome the limitations encountered by single actuation applications. Moreover, the magnetic particles offer other functions such as removing organic pollutants via the Fenton reaction. Janus-structured motors provide a broad perspective not only for biosensing, optical detection, and on-chip separation applications but also for environmental water treatment due to the catalytic activities of multistimulus-responsive micromotors.

Asymmetric geometry (aspect ratio >1), moderate stiffness (i.e., semielasticity), large surface area, and low mucoadhesion of nanoparticles are the main features to reach the brain by penetrating across the nasal mucosa. Herein, a new application has been presented for the use of multifunctional Janus nanoparticles (JNPs) with controllable geometry and size as a nose-to-brain (N2B) delivery system by changing proportions of Precirol ATO 5 and polycaprolactone compartments and other operating conditions. To bring to light the N2B application of JNPs, the results are presented in comparison with polymer and solid lipid nanoparticles, which are frequently used in the literature regarding their biopharmaceutical aspects: mucoadhesion and permeability through the nasal mucosa. The morphology and geometry of JPs were observed via cryogenic-temperature transmission electron microscopy images, and their particle sizes were verified by dynamic light scattering, atomic force microscopy, and scanning electron microscopy. Although all NPs showed penetration across the mucus barrier, the best increase in penetration was observed with asymmetric and semielastic JNPs, which have low interaction ability with the mucus layer. This study presents a new and promising field of application for a multifunctional system suitable for N2B delivery, potentially benefiting the treatment of brain tumors and other central nervous system diseases.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy., (© 2023 Altas et al.)

Cas9 targets genomic loci with high specificity. For knockin with double-strand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. In this study, we demonstrate a high-throughput workflow to comparatively assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations, yielding increases in knockin efficiency and precision. Cas9-RC, a novel fusion Cas9 flanked by eRad18 and CtIP [HE] , increased knockin performance in vitro and in vivo in the developing mouse brain. Continued comparative assessment of editing efficiency and precision with this framework will further the development of high-performance editing agents for in vivo knockin and future genome therapeutics.

The kinase mTOR is a signaling hub for pathways that regulate cellular growth. In neurons, the subcellular localization of mTOR takes on increased significance. Here, we review findings on the localization of mTOR in axons and offer a perspective on how these may impact our understanding of nervous system development, function, and disease. We propose a model where mTOR accumulates in local foci we term mTOR outposts, which can be found in processes distant from a neuron's cell body. In this model, pathways that funnel through mTOR are gated by local outposts to spatially select and amplify local signaling. The presence or absence of mTOR outposts in a segment of axon or dendrite may determine whether regional mTOR-dependent signals, such as nutrient and growth factor signaling, register toward neuron-wide responses. In this perspective, we present the emerging evidence for mTOR outposts in neurons, their putative roles as spatial gatekeepers of signaling inputs, and the implications of the mTOR outpost model for neuronal protein synthesis, signal transduction, and synaptic plasticity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Atlas, Romanowski, Bunce and Poulopoulos.)

Kaufman oculocerebrofacial syndrome (KOS) is a severe autosomal recessive disorder characterized by intellectual disability, developmental delays, microcephaly, and characteristic dysmorphisms. Biallelic mutations of UBE3B, encoding for a ubiquitin ligase E3B are causative for KOS. In this report, we characterize neuronal functions of its murine ortholog Ube3b and show that Ube3b regulates dendritic branching in a cell-autonomous manner. Moreover, Ube3b knockout (KO) neurons exhibit increased density and aberrant morphology of dendritic spines, altered synaptic physiology, and changes in hippocampal circuit activity. Dorsal forebrain-specific Ube3b KO animals show impaired spatial learning, altered social interactions, and repetitive behaviors. We further demonstrate that Ube3b ubiquitinates the catalytic γ-subunit of calcineurin, Ppp3cc, the overexpression of which phenocopies Ube3b loss with regard to dendritic spine density. This work provides insights into the molecular pathologies underlying intellectual disability-like phenotypes in a genetically engineered mouse model., (© 2020. The Author(s), under exclusive licence to Springer Nature Limited.)

Although similar in molecular composition, synapses can exhibit strikingly distinct functional transmitter release and plasticity characteristics. To determine whether ultrastructural differences co-define this functional heterogeneity, we combine hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to compare two functionally distinct synapses: hippocampal Schaffer collateral and mossy fiber synapses. We find that mossy fiber synapses, which exhibit a lower release probability and stronger short-term facilitation than Schaffer collateral synapses, harbor lower numbers of docked synaptic vesicles at active zones and a second pool of possibly tethered vesicles in their vicinity. Our data indicate that differences in the ratio of docked versus tethered vesicles at active zones contribute to distinct functional characteristics of synapses., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the transcription factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9-[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Munc13 catalyzes the transit of syntaxin from a closed complex with Munc18 into the ternary SNARE complex. Here we report a new function of Munc13, independent of Munc18: it promotes the proper syntaxin/synaptobrevin subconfiguration during assembly of the ternary SNARE complex. In cooperation with Munc18, Munc13 additionally ensures the proper syntaxin/SNAP-25 subconfiguration. In a reconstituted fusion assay with SNAREs, complexin, and synaptotagmin, inclusion of both Munc13 and Munc18 quadruples the Ca 2+ -triggered amplitude and achieves Ca 2+ sensitivity at near-physiological concentrations. In Munc13-1/2 double-knockout neurons, expression of a constitutively open mutant of syntaxin could only minimally restore neurotransmitter release relative to Munc13-1 rescue. Together, the physiological functions of Munc13 may be related to regulation of proper SNARE complex assembly., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dendritic spines are the major transmitter reception compartments of glutamatergic synapses in most principal neurons of the mammalian brain and play a key role in the function of nerve cell circuits. The formation of functional spine synapses is thought to be critically dependent on presynaptic glutamatergic signaling. By analyzing CA1 pyramidal neurons in mutant hippocampal slice cultures that are essentially devoid of presynaptic transmitter release, we demonstrate that the formation and maintenance of dendrites and functional spines are independent of synaptic glutamate release., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The chemical synapse displays specialized intercellular adhesion between pre- and potsynaptic plasma membranes mediated by synaptic cell adhesion proteins. In this asymmetric cell adhesion, pre- and postsynapses have their own unique functions; the presynaptic terminal releases neurotransmitter, which diffuses through the synaptic cleft and is received by receptors accumulated at the postsynapse. Such distinct modes of actions of pre- and postsynapses in synaptic neurotransmission are the rate-limiting factors in signal processing in the brain, and thus protein-protein interactions within the pre- and postsynaptic scaffold are of particular importance for brain function by regulating the pre- and postsynaptic function. In the present paper, we outline a method to screen for binding partners of synaptic scaffold proteins biochemically.

Parkinson's disease (PD) is a disorder characterized by the degeneration of certain neuronal populations in the central and peripheral nervous system. One of the hallmarks of the disease is the toxic accumulation of proteins within susceptible neurons due to major impairment in the degradation/clearance protein systems.RTP801 is a pro-apoptotic protein that is sufficient and necessary to induce neuronal death in cellular and animal models of PD. RTP801 is also upregulated in sporadic and parkin mutant PD brains. Here, we report the role of NEDD4, an E3 ligase involved in α-synuclein degradation and PD pathogenesis, in the regulation of RTP801 protein levels and toxicity. NEDD4 polyubiquitinates RTP801 in a cell-free system and in cellular cultures, and they interact physically. NEDD4 conjugates K63-ubiquitin chains to RTP801 and targets it for degradation. NEDD4 regulates RTP801 protein levels in both cultured cells and in the brain tissue. NEDD4 levels are diminished in nigral neurons from human PD brains. Interestingly, neurotoxin 6-OHDA decreases dramatically NEDD4 protein expression but elevates RTP801 protein levels. Moreover, NEDD4 protects neuronal PC12 cells from both 6-OHDA and RTP801-induced toxicity. In primary cortical neurons, NEDD4 knockdown toxicity is mediated by RTP801 since the double knockdown of RTP801 and NEDD4 abrogates the loss of phospho Ser473-Akt and the appearance of caspase-cleaved spectrin fragments.Thus, NEDD4 ligase regulates RTP801 and is sensitive to PD-associated oxidative stress. This suggests that NEDD4 loss of function in PD could contribute importantly into neuronal death by elevating RTP801.

Erythropoietin (EPO) exerts potent neuroprotective, neuroregenerative and procognitive functions. However, unequivocal demonstration of erythropoietin receptor (EPOR) expression in brain cells has remained difficult since previously available anti-EPOR antibodies (EPOR-AB) were unspecific. We report here a new, highly specific, polyclonal rabbit EPOR-AB directed against different epitopes in the cytoplasmic tail of human and murine EPOR and its characterization by mass spectrometric analysis of immuno-precipitated endogenous EPOR, Western blotting, immunostaining and flow cytometry. Among others, we applied genetic strategies including overexpression, Lentivirus-mediated conditional knockout of EpoR and tagged proteins, both on cultured cells and tissue sections, as well as intracortical implantation of EPOR -transduced cells to verify specificity. We show examples of EPOR expression in neurons, oligodendroglia, astrocytes and microglia. Employing this new EPOR-AB with double-labeling strategies, we demonstrate membrane expression of EPOR as well as its localization in intracellular compartments such as the Golgi apparatus. Moreover, we show injury-induced expression of EPOR. In mice, a stereotactically applied stab wound to the motor cortex leads to distinct EpoR expression by reactive GFAP-expressing cells in the lesion vicinity. In a patient suffering from epilepsy, neurons and oligodendrocytes of the hippocampus strongly express EPOR. To conclude, this new analytical tool will allow neuroscientists to pinpoint EPOR expression in cells of the nervous system and to better understand its role in healthy conditions, including brain development, as well as under pathological circumstances, such as upregulation upon distress and injury.

The aim of this study was to evaluate the relationship between the maxillary sinus volumes and the nasal septal deviation angles in patients with antrochoanal polyps (ACP). 76 patients who underwent ACP surgery were included in the study. Of those 36 patients who had multislice computed tomography (MSCT) were evaluated to calculate maxillary sinus volume. The records of paranasal MSCT of 36 healthy people without any paranasal sinus diseases or surgery constituted age- and gender-matched healthy controls. Maxillary sinüs volumes and septal deviation angles were calculated using the paranasal MSCT volume-rendering technique. Thirty-six patients in the ACP group were compared with 36 polyp side-matched healthy people. The mean age was 16.6 ± 6.7 years in both groups. Statistically, the mean value of the maxillary sinus volume was significantly higher in the ACP group compared with the ACP side-matched control group (15.1 ± 4.6 versus 12.0 ± 3.5 mm(3)) (p = 0.002). Furthermore, the mean value of the maxillary sinus volume in the non-polyp side (14.2 ± 4.7 mm(3)) was statistically higher in the ACP group compared with the side-matched control group volume (11.9 ± 3.8 mm(3)) (p = 0.024). In addition, Fifty-three of 76 ACP patients had septal deviation. While the septal deviation was on the same side with the ACP in 17 patients, it was on the opposite side in 36 patients. In conclusion, the maxillary sinus volumes increased in ACP patients compared with the healthy control group. Many patients had nasal septal deviation on the opposite side of the ACP.

Brain-derived neurotrophic factor (BDNF) is widely reported to enhance synaptic vesicle (SV) exocytosis and neurotransmitter release. But it is still unclear whether BDNF enhances SV recycling at excitatory terminals only, or at both excitatory and inhibitory terminals. In the present study, in a direct comparison using cultured rat hippocampal neurons, we demonstrate that BDNF enhances both spontaneous and activity-dependent neurotransmitter release from excitatory terminals, but not from inhibitory terminals. BDNF treatment for 5 min or 48 h increased both spontaneous and activity-induced anti-synaptotagmin1 (SYT1) antibody uptake at excitatory terminals marked with vGluT1. Conversely, BDNF treatment did not enhance spontaneous or activity-induced uptake of anti-SYT1 antibodies in inhibitory terminals marked with vGAT. Time-lapse imaging of FM1-43 dye destaining in excitatory and inhibitory terminals visualized by post-hoc immunostaining of vGluT1 and vGAT also showed the same result: The rate of spontaneous and activity-induced destaining was increased by BDNF at excitatory synapses, but not at inhibitory synapses. These data demonstrate that BDNF enhances SV exocytosis in excitatory but not inhibitory terminals. Moreover, BDNF enhanced evoked SV exocytosis, even if vesicles were loaded under spontaneous vesicle recycling conditions. Thus, BDNF enhances both spontaneous and activity-dependent neurotransmitter release on both short and long time-scales, by the same mechanism.

Objective: Pharyngocutaneous fistula (PCF) is the most common postoperative complication after total laryngectomy. The aim of this study was to determine the relationship between PCF with pharyngeal repair time and the number of mucosal sutures., Methods: The medical records of 47 patients who underwent total laryngectomy were assessed prospectively. The pharyngeal repair time and the number of horizontal, vertical, and cricopharyngeal muscle sutures were recorded. We observed the occurrence of PCF in 14 patients (29.8%). The mean time for pharyngeal repair in the PCF group was 22 minutes 59 seconds ± 5 minutes, and the mean number of vertical, horizontal, and cricopharyngeal muscle sutures was 8.57 ± 2.6, 11.14 ± 2.1, and 8.45 ± 1.9, respectively. The mean time for pharyngeal repair in the non-PCF group was 22 minutes 21 seconds ± 5 minutes, and the mean number of vertical, horizontal, and cricopharyngeal muscle sutures was 9.54 ± 2.6, 10.84 ± 2.3, and 7.36 ± 2.7, respectively. The differences in the pharyngeal repair time and the number of vertical, horizontal, and cricopharyngeal sutures between the 2 groups were not statistically significant (P > 0.05). Besides, there was no statistically significant difference between the mean suture counts per centimeter in horizontal plane (1.46 ± 0.35) and vertical plane (0.98 ± 0.25) in the PCF group and the mean suture counts per centimeter in horizontal plane (1.42 ± 0.37) and vertical plane (1.13 ± 0.3) in the non-PCF group (P > 0.05)., Conclusions: There was no relation between PCF development with pharyngeal repair time, mucosal suture count, and suture frequency after total laryngectomy.

The purpose of our study was to apply the new classification criteria proposed by the Acute Kidney Injury Network (AKIN) in a pediatric population and to determine the clinical characteristics, laboratory features and outcomes of acute kidney injury (AKI) in a tertiary pediatric nephrology center in Turkey. Patients' charts from January 2003 to August 2008 were retrospectively evaluated. One hundred patients (55 male; 45 female) were enrolled. Median age at the time of AKI was 7 years (range 1 month-18 years). Patients' AKI was classified according to the staging system as follows: 25% stage 1, 36% stage 2 and 39% stage 3. The etiology of AKI was bone marrow transplantation related in 27%, renal disease in 14%, dehydration in 10%, nephrotoxic medication in 8%, cardiac surgery related in 8%, and congenital anomalies in 2%. Multiple etiologic factors with underlying chronic diseases were present in 31% of the patients. Dialysis was needed in 45% of the patients. Mortality rate was 33%. Dialysis need and mortality rate were higher in stage 2 and stage 3 patients, with a more favorable prognosis in stage 1 patients. Mortality rate was higher in patients that had undergone cardiac surgery and in those with multiple etiologic factors. The proposed AKIN staging successfully reflected the course of patients with AKI. The underlying cause of AKI seemed to be an important risk factor for death.

Purpose: To determine reirradiation results of patients with recurrent non-metastatic non-small cell lung cancer (NSCLC)., Patients and Methods: 38 NSCLC patients who showed clinical and/or radiological progression and were retreated with hypofractionated irradiation (RT) were retrospectively evaluated. Two parallel or oblique opposed fields were used for reirradiation of the recurrent tumor while excluding the spinal cord. "Improvement" and "complete or near complete response" were defined as > or = 50% and 75-100% regression of symptoms, respectively. Log-rank test, chi-square test and Cox regression analysis were used for statistical analyses., Results: Median age was 58 years (range 33-80) and only 3 patients were females. Median follow-up was 13.5 months (range 4-65). In the initial and second course of RT the total dose was 30 Gy (range 28.8-67.2) and 25 Gy (range 5-30) and the number of fractions was 10 (range 9-33) and 10 (range 1-10), respectively. The median interval between the two RT courses was 35 weeks (range 4-189). After reirradiation improvement was observed in 86% of the patients assessable for hemoptysis, in 77% with cough, in 69% with dyspnea, and in 60% with thoracic pain. After reirradiation, the median survival time was 3 months (range 0-55). Two-year survival rates from diagnosis were 28.8% and from reirradiation 5.8%. An interval more than 35 weeks between the end of initial RT and the start of reirradiation was found as the only independent prognostic factor affecting survival. No grade III-IV RTOG late side effects were observed., Conclusion: In initially non-metastatic NSCLC patients, reirradiation can be a safe and effective treatment for palliation after recurrence. Large prospective studies are needed to confirm the safety, effectiveness and economical advantages of this modality.