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Neurite outgrowth is a fundamental process in neurons that produces extensions and, consequently, neural connectivity. Neurite damage and atrophy are observed in various brain injuries and disorders. Understanding the intrinsic pathways of neurite outgrowth is essential for developing strategies to stimulate neurite regeneration. Insulin is a pivotal hormone in the regulation of glucose homeostasis. There is increasing evidence for the neurotrophic functions of insulin, including the induction of neurite outgrowth. However, the associated mechanism remains elusive. Here, we demonstrate that insulin potentiates neurite outgrowth mediated by the small GTPases ADP-ribosylation factor 6 (ARF6) and Ras-related C3 botulinum toxin substrate 1 (Rac1) through the neuronal adaptor FE65. Moreover, insulin enhances atypical protein kinase Cι/λ (PKCι/λ) activation and FE65 phosphorylation at serine 459 (S459) in neurons and mouse brains. In vitro and cellular assays show that PKCι/λ phosphorylated FE65 at S459. Consistently, insulin potentiates FE65 S459 phosphorylation only in the presence of PKCι/λ. Phosphomimetic studies show that an FE65 S459E mutant potently activates ARF6, Rac1, and neurite outgrowth. Notably, this phosphomimetic mutation enhances the FE65-ARF6 interaction, a process that promotes ARF6-Rac1-mediated neurite outgrowth. Likewise, insulin treatment and PKCι/λ overexpression potentiate the FE65-ARF6 interaction. Conversely, PKCι/λ knockdown suppresses the stimulatory effect of FE65 on ARF6-Rac1-mediated neurite outgrowth. The effect of insulin on neurite outgrowth is also markedly attenuated in PKCι/λ knockdown neurons, in the presence and absence of FE65. Our findings reveal a novel mechanism linking insulin with ARF6-Rac1-dependent neurite extension through the PKCι/λ-mediated phosphorylation of FE65., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Myelin-associated glycoprotein (MAG) and Nogo inhibit neurite outgrowth by binding to receptors such as NgR1, PirB and LRP1, and they have also been shown to induce phosphorylation of Smad2, a key intermediate in the transforming growth factor β (TGFβ) signalling pathway. In this study, we determined that MAG and Nogo do not transactivate the TGFβ receptor through their canonical receptors or discoidin domain receptor 1, which we identified as a novel receptor for MAG and Nogo. Instead, MAG and Nogo promoted Smad2 phosphorylation by stimulating secretion of TGFβ. Proteomic analysis of the neuronal secretome revealed that MAG also regulated the secretion of proteins that affect central nervous system plasticity-inducing the secretion of S100A6, septin-7 and neurofascin 186, while inhibiting the secretion of frataxin, MAP6, syntenin-1 and GAP-43. This represents a novel function for MAG that has broad implications for the treatment for spinal cord injury., (© 2022 Federation of European Biochemical Societies.)

Asymmetric subcellular mRNA localization allows spatial regulation of gene expression and functional compartmentalization. In neurons, localization of specific mRNAs to neurites is essential for cellular functioning. However, it is largely unknown how transcript sorting works in a sequence-specific manner. Here, we combined subcellular transcriptomics and massively parallel reporter assays and tested ∼50 000 sequences for their ability to localize to neurites. Mapping the localization potential of >300 genes revealed two ways neurite targeting can be achieved: focused localization motifs and broadly encoded localization potential. We characterized the interplay between RNA stability and localization and identified motifs able to bias localization towards neurite or soma as well as the trans-acting factors required for their action. Based on our data, we devised machine learning models that were able to predict the localization behavior of novel reporter sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native full-length transcripts., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neurite outgrowth is an integrated whole cell response triggered by the cannabinoid-1 receptor. We sought to identify the many different biochemical pathways that contribute to this whole cell response. To understand underlying mechanisms, we identified subcellular processes (SCPs) composed of one or more biochemical pathways and their interactions required for this response. Differentially expressed genes and proteins were obtained from bulk transcriptomics and proteomic analysis of extracts from cells stimulated with a cannabinoid-1 receptor agonist. We used these differentially expressed genes and proteins to build networks of interacting SCPs by combining the expression data with prior pathway knowledge. From these SCP networks, we identified additional genes that when ablated, experimentally validated the SCP involvement in neurite outgrowth. Our experiments and informatics modeling allowed us to identify diverse SCPs such as those involved in pyrimidine metabolism, lipid biosynthesis, and mRNA splicing and stability, along with more predictable SCPs such as membrane vesicle transport and microtubule dynamics. We find that SCPs required for neurite outgrowth are widely distributed among many biochemical pathways required for constitutive cellular functions, several of which are termed 'deep', since they are distal to signaling pathways and the key SCPs directly involved in extension of the neurite. In contrast, 'proximal' SCPs are involved in microtubule growth and membrane vesicle transport dynamics required for neurite outgrowth. From these bioinformatics and dynamical models based on experimental data, we conclude that receptor-mediated regulation of subcellular functions for neurite outgrowth is both distributed, that is, involves many different biochemical pathways, and deep., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Spinal cord injury (SCI) is the most severe result of spine injury, but no effective therapy exists to treat SCI. We have previously shown that the E3 ubiquitin ligase Two RING fingers and DRIL 1 (Triad1) promotes neurite outgrowth after SCI. However, the mechanism by which Triad1 affects neuron growth and the potential involvement of its ubiquitination activity is unclear. Neuroprotective cytokine pleiotrophin (PTN) can promote microglia proliferation and neurotrophic factor secretion to achieve neuroprotection. We find using immunostaining and behavioral assays in rats that the expression of Triad1 and the PTN was peaked at 1 day after SCI and Triad1 improved motor function and histomorphological injury after SCI. We show using flow cytometry and astrocyte/neuronal coculture assays that Triad1 overexpression promoted PTN protein levels, neurotrophic growth factor (NGF) expression, brain-derived neurotrophic factor (BDNF) expression, astrocyte and neuronal viability, and neurite outgrowth but suppressed astrocyte apoptosis, while shRNA-mediated knockdown of Triad1 and PTN had the opposite effects. Ubiquitin ligase murine double mutant 2 (MDM2) has previously been demonstrated to participate in the process of neurite outgrowth and mediate ubiquitination of p53. Furthermore, we demonstrate overexpression of MDM2 downregulated PTN protein levels, NGF expression and BDNF expression in astrocytes, and inhibited neurite outgrowth of neurons. In addition, MDM2 facilitated PTN ubiquitination, which was reversed by Triad1. Finally, we show simultaneous sh-PTN and MDM2 overexpression attenuated the neurite outgrowth-promoting effect of Triad1 overexpression. In conclusion, we propose Triad1 promotes astrocyte-dependent neurite outgrowth to accelerate recovery after SCI by inhibiting MDM2-mediated PTN ubiquitination., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) exhibits systemic impacts on the metabolism, while metabolic alteration contributes to the risk and progression of PD. Bile acids (BA) metabolism disturbance has been linked to PD pathology. Membrane-bound G protein-coupled bile acid receptor 1 (GPBAR1) is expressed in the brain and thought to be neuroprotective; however, the role of GPBAR1 in PD remains unknown. The current study aimed to explore the effect of GPBAR1 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice with dopaminergic (DA) neuron-specific Gpbar1 knockdown or central GPBAR1 activation. The underlying mechanisms were investigated using mesencephalic primary neurons analyzed. Our study found that GPBAR1 was reduced in the substantia nigra of PD patients and MPTP-PD mice, and its expression was negatively correlated with the severity of PD-related features. Genetic downregulation of Gpbar1 in mouse mesencephalic DA neurons exacerbated MPTP-induced neurobehavioral and neuropathological deficits, whereas activation of central GPBAR1 with INT-777 (INT) relieved it. Moreover, in vivo and in vitro experiments showed the neurite- and synapse-protective effects of GPBAR1 activation in PD model. Mechanistically, by promoting the nuclear localization of cohesin subunit RAD21, GPBAR1 activation increased opioid-binding cell adhesion molecule (Opcml) expression, thereby inhibiting neurite and synapse degeneration of DA neurons in PD model. Collectively, our findings demonstrate that GPBAR1 is implicated in PD pathogenesis and activation of central GPBAR1 with INT antagonizes neurodegenerative pathology in PD model. This neuroprotection, at least in part, is attributed to the RAD21-OPCML signaling in neurons. Hence, GPBAR1 may serve as a promising candidate target for PD treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Alzheimer's Disease (AD) affects millions globally, but therapy development is lagging. New experimental systems that monitor neuronal functions in conditions approximating the AD brain may be beneficial for identifying new therapeutic strategies., Methods: We expose cultured neurons to aqueous-soluble human brain extract from 43 individuals across a spectrum of AD pathology. Multi-electrode arrays (MEAs) and live-cell imaging were used to assess neuronal firing and neurite integrity (NI), respectively, following treatments of rat cortical neurons (MEA) and human iPSC-derived neurons (iN) with human brain extracts., Results: We observe associations between spontaneous activity and Aβ42:40 levels, between neurite integrity and oligomeric Aβ, and between neurite integrity and tau levels present in the brain extracts. However, these associations with Aβ and tau do not fully account for the effects observed. Proteomic profiling of the brain extracts revealed additional candidates correlated with neuronal structure and activity. Neurotoxicity in MEA and NI assays was associated with proteins implicated in lysosomal storage disorders, while neuroprotection was associated with proteins of the WAVE regulatory complex controlling actin cytoskeleton dynamics. Elevated ganglioside GM2 activator (GM2A) associates with reductions in both NI and MEA activity, and cell-derived GM2A alone is sufficient to induce a loss of neurite integrity and a reduction in neuronal firing., Conclusions: The techniques and data herein introduce a system for modeling neuronal vulnerability in response to factors in the human brain and provide insights into proteins potentially contributing to AD pathogenesis., (© 2022. The Author(s).)

The lack of supportive Schwann cells in segmental nerve lesions seems to be one cornerstone for the problem of insufficient nerve regeneration. Lately, adipose-tissue-derived stem cells (ASCs) differentiated towards SC (Schwann cell)-like cells seem to fulfill some of the needs for ameliorated nerve recovery. In this study, three differentiation protocols were investigated for their ability to differentiate ASCs from rats into specialized SC phenotypes. The differentiated ASCs (dASCs) were compared for their expressions of neurotrophins (NGF, GDNF, BDNF), myelin markers (MBP, P0), as well as glial-marker proteins (S100, GFAP) by RT-PCR, ELISA, and Western blot. Additionally, the influence of the medium conditioned by dASCs on a neuron-like cell line was evaluated. The dASCs were highly diverse in their expression profiles. One protocol yielded relatively high expression rates of neurotrophins, whereas another protocol induced myelin-marker expression. These results were reproducible when the ASCs were differentiated on surfaces potentially used for nerve guidance conduits. The NGF secretion affected the neurite outgrowth significantly. It remains uncertain what features of these SC-like cells contribute the most to adequate functional recovery during the different phases of nerve recovery. Nevertheless, therapeutic applications should consider these diverse phenotypes as a potential approach for stem-cell-based nerve-injury treatment.

When the regulation of axonal and dendritic growth is altered, the neuronal network becomes disordered, which may contribute to the development of psychiatric disorders. Some genome analyses have suggested relationships between mutations in strawberry notch homologue 1 (SBNO1) and neurodevelopmental disorders. However, the function of SBNO1 has not yet been reported. Here, SBNO1 expression pattern during the development of the cerebral cortex in mice was examined. SBNO1 was strongly expressed in the cortical plate and its expression was maintained at a low level during the postnatal stage. CRISPR/Cas9-based knockout of Sbno1 in Neuro2A cultured cells showed delayed growth of neurites. A cortical neuron-specific conditional knockout mouse was constructed, which resulted in hypotrophy of axon bundles and dendrites in cortical neurons. Thus, when mutated, SBNO1 is a candidate gene for psychiatric diseases, such as schizophrenia, as suggested by human genome studies., (© 2022 Japanese Society of Developmental Biologists.)

Three rare spirocyclohexadienone-type neolignans, magnoflorins A-C (1-3), and three known analogs (4-6), were isolated from the leaves of Magnolia liliiflora. Magnoflorin D (4) was obtained from natural resources for the first time. The chemical structures and absolute configurations of 1-4 were elucidated through detailed analysis of HR-ESI-MS, IR, 1 H, 13 C, and 2D NMR, and ECD experiments. The absolute configuration of 5 were characterized by X-ray crystallography in present study. Moreover, compounds 4 and 5 displayed moderate neuroprotective activity against corticosterone-induced PC12 cells injury at 20 μM with cell viability of 71.5±0.99 % and 73.0±1.42 %, respectively, compared to the model group with 60.83±0.93 %. Compound 6 could enhance neurite outgrowth of nerve growth factor (NGF)-induced PC12 cells at 10 μM with the differentiation rate of 11.98 %, compared with 20.49 % of 50 ng/ml NGF., (© 2022 Wiley-VHCA AG, Zurich, Switzerland.)

Parkinson's disease is characterized by the deposition of α-synuclein, which leads to synaptic dysfunction, the loss of neuronal connections and ultimately progressive neurodegeneration. Despite extensive research into Parkinson's disease pathogenesis, the mechanisms underlying α-synuclein-mediated synaptopathy have remained elusive. Several lines of evidence suggest that altered nicotinamide adenine dinucleotide (NAD+) metabolism might be causally related to synucleinopathies, including Parkinson's disease. NAD+ metabolism is central to the maintenance of synaptic structure and function. Its synthesis is mediated by nicotinamide mononucleotide adenylyltransferases (NMNATs), but their role in Parkinson's disease is not known. Here we report significantly decreased levels of NMNAT3 protein in the caudate nucleus of patients who have died with Parkinson's disease, which inversely correlated with the amount of monomeric α-synuclein. The detected alterations were specific and significant as the expression levels of NMNAT1, NMNAT2 and sterile alpha and TIR motif containing 1 (SARM1) were not significantly different in Parkinson's disease patients compared to controls. To test the functional significance of these findings, we ectopically expressed wild-type α-synuclein in retinoic acid-differentiated dopaminergic SH-SY5Y cells that resulted in decreased levels of NMNAT3 protein plus a neurite pathology, which could be rescued by FK866, an inhibitor of nicotinamide phosphoribosyltransferase that acts as a key enzyme in the regulation of NAD+ synthesis. Our results establish, for the first time, NMNAT3 alterations in Parkinson's disease and demonstrate in human cells that this phenotype together with neurite pathology is causally related to α-synucleinopathy. These findings identify alterations in the NAD+ biosynthetic pathway as a pathogenic mechanism underlying α-synuclein-mediated synaptopathy., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Alpha synuclein (aSyn) and its aggregation are crucial for the neurodegeneration of Parkinson's disease (PD). aSyn was initially described in the nucleus and presynaptic nerve terminals. However, the biology of nuclear aSyn and the link of aSyn between subcellular compartments are less understood. Current knowledge suggests the existence of various aSyn species with distinct structural and biochemical properties. Here, we identified a C-terminal-targeting aSyn antibody (Nu-aSyn-C), which has a high immunoaffinity towards aSyn in the nucleus. Comparing the Nu-aSyn-C antibody to aSyn antibodies developed against phosphorylated or aggregated forms, we observed that nuclear aSyn differs from cytosolic aSyn by an increased phosphorylation and assembly level in proliferating cells. Employing Nu-aSyn-C, we characterized aSyn distribution during neuronal differentiation in midbrain dopaminergic neurons (mDANs) derived from human-induced pluripotent stem cells (hiPSCs) and Lund human mesencephalic cells, and in primary rat hippocampal neurons. We detected a specific translocation pattern of aSyn during neuronal differentiation from the nucleus to the soma and finally to neuronal processes. Interestingly, a remarkable shift of Nu-aSyn-C-positive species towards neurites was detected in hiPSC mDANs from a PD patient carrying aSyn gene duplication. Together, our results reveal distinct nuclear and cytosolic aSyn species that redistribute during neuronal differentiation-a process that is altered in PD-derived neurons.

Objectives: Electrophysiological studies have revealed that abnormal function of the corticospinal pathway might contribute to chronic ankle instability, but structural evidence underlying the abnormality is lacking. The purpose of this study was to quantitate microstructural differences between corticospinal tracts in patients with chronic ankle instability and healthy controls., Design: Cross-sectional., Methods: Seventeen patients with chronic ankle instability and sixteen healthy controls underwent diffusion weighted-imaging scans using an ultra-high-field 7.0 Tesla magnetic resonance imaging scanner. We focused on corticospinal tracts as a region of interest and performed classical diffusion tensor imaging and the advanced neurite orientation dispersion and density imaging outcomes that measured the microstructure of white matter tracts. Correlation analyses were also performed between the significantly different diffusion outcomes in both groups., Results: The patients with chronic ankle instability showed significantly lower fractional anisotropy (p-corrected = 0.045) and higher orientation dispersion index (p-corrected = 0.033) when compared with healthy controls. These two measures were significantly correlated in the healthy controls (r = -0.56, p = 0.024) and the CAI patient group (r = -0.53, p = 0.029)., Conclusions: This study revealed that the contralateral corticospinal tract of the unstable ankle in patients with chronic ankle instability exhibited impaired integrity, which was associated with abnormally organized neurites. We propose that this is a useful target for the clinical assessment of chronic ankle instability and the development of targeted neuromuscular rehabilitation., (Copyright © 2022 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.)

The effects of microRNA-29a-3p in the proliferation process of nerve cells are unclear. The purpose of this study is to delve into the regulatory role of microRNA-29a-3p, via interaction with phosphatase and tension homolog (PTEN), in the SH-SY5Y cell proliferation process. Different expressions of microRNA-29a-3p in the SH-SY5Y cells were constructed by transfected miRNA-29a-3p mimic and inhibitor. The effects of cell transfection and the mRNA expressions of PTEN, Akt, and mTOR were detected by qPCR. The expressions of PTEN, Akt, and mTOR protein and the phosphorylation levels of Akt and mTOR were examined using Western blotting. Nerve cell proliferation activity and neurite length of each group were measured and examined by the use of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2Htetrazolium bromide (MTT), and morphological examination. We observed that the levels of PTEN mRNA and protein were distinctly decreased in the microRNA-29a-3p mimic group, but the expressions of the phosphorylated Akt and mTOR mRNA and protein were distinctly upregulated. In the transfected miRNA-29a-3p inhibitor SH-SY5Y cells, the expressions of miRNA-29a-3p were significantly suppressed; however, the expressions of PTEN gene and protein were significantly enhanced. The expressions of phosphorylated Akt and mTOR in the downregulated microRNA-29a-3p group distinctly were suppressed. The SH-SY5Y cell proliferation activity and neurite length in the upregulated microRNA-29a-3p group increased significantly. Our findings revealed that microRNA-29a-3p could enhance the proliferation activity of SH-SY5Y cells and promote neurite growth by inhibiting the expression of PTEN and regulating PI3K/Akt/mTOR signaling pathway., Competing Interests: All the authors declare no conflict of interests., (Copyright © 2022 Wansheng Gao et al.)

The hM4Di receptor-based chemogenetic DREADD system has been widely used to suppress neuronal activities, which has contributed substantially to the identification of behavior-associated neuronal circuitries including those in the striatum. One major mechanism by which hM4Di receptor activation suppresses neuronal activity is that the activation reduces membrane excitability, which is thought to be mediated by the opening of GIRK channels. However, previous studies have suggested that GIRK channels are barely expressed in the striatum, which naturally raises the question whether the hM4Di receptor activation-induced reduction in membrane excitability found in striatal medium spiny neurons (MSNs, which constitute 95-98% of the striatal neuronal population) is truly mediated by the endogenous GIRK channels in such scarcity. This study aims to answer this question by applying a GIRK channel-selective blocker, tertiapin-Q (TPNQ), to striatal MSNs. This study first verified that application of clozapine (CZP), an hM4Di receptor agonist, to MSNs expressing the hM4Di receptors hyperpolarized the cell membrane, and reduced membrane excitability and input resistance. This study next revealed that TPNQ post-treatment completely canceled the above CZP-induced electrophysiological effects and that TPNQ pretreatment mostly prevented further expression of the above CZP-induced electrophysiological effects. In addition, confocal microscopy imaging also revealed significant above-background GIRK1 immunofluorescence signals in striatal MSNs. These data suggest that the TPNQ-sensitive GIRK channels, despite being expressed at low levels, are likely the major mediator downstream of hM4Di receptor activation to reduce membrane excitability in striatal MSNs. These results imply that the notion held by scientists in the field that GIRK channels are absent in the striatum or their expression level is not significant enough to exert any function might be oversimplified or incorrect.

Prostaglandin E2 (PGE2) is a membrane-derived lipid signaling molecule important in neuronal development. Abnormal levels of PGE2, due to environmental insults prenatal development, have been linked to brain pathologies. We have previously shown that the addition of PGE2 to neuroectodermal (NE4C) stem cells affects early stages of neuronal differentiation (day 0-8) including increased stem cell motility, accelerated formation of neurospheres, and elevated calcium levels in growth cones. In this study, we further examine whether PGE2 can influence actin-dependent neuronal morphology in later stages (day 8-12) of NE4C cell differentiation. We show that exposure to PGE2 from the initiation of differentiation increased neurite length and the proportion of neurites that formed axonal loops. We also observed changes in the proportion of turning growth cones as the differentiation progressed, with a reduced likelihood of observing turning (or asymmetrical) growth cones on day 8 and increased odds on days 10 and 12. Moreover, we showed for the first time that the observed changes in cytoskeletal morphology were PGE2/PKA dependent. Interestingly, we also found that PGE2 decreased the total protein levels of the actin-bound form of spinophilin and increased levels of unbound PKA-phosphorylated ser94-spinophilin. Hence, we propose that exposure to PGE2 can destabilize the actin cytoskeleton at various stages of neuronal differentiation due to dissociation of ser94-spinophilin causing changes in neuronal morphology., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Alzheimer's disease (AD) is characterized by amyloid plaques and neurofibrillary tangles accompanied by progressive neurite loss. Mitochondria play pivotal roles in AD development. PRDX3 is a mitochondrial peroxide reductase critical for H 2 O 2 scavenging and signal transduction. In this study, we found that PRDX3 knockdown (KD) in the N2a-APPswe cell line promoted retinoic acid (RA)-induced neurite outgrowth but did not reduce the viability of cells damaged by tert-butyl hydroperoxide (TBHP). We found that knocking down PRDX3 expression induced dysregulation of more than one hundred proteins, as determined by tandem mass tag (TMT)-labeled proteomics. A Gene Ontology (GO) analysis revealed that the dysregulated proteins were enriched in protein localization to the plasma membrane, the lipid catabolic process, and intermediate filament cytoskeleton organization. A STRING analysis showed close protein-protein interactions among dysregulated proteins. The expression of Annexin A1 (ANXA1), serine (Ser)-/threonine (Thr)-protein phosphatase 2A catalytic subunit alpha isoform (PP2A) and glutathione S-transferase Mu 2 (GSTM2) was significantly upregulated in PRDX3-KD N2a-APPswe cell lines, as verified by western blotting. Our study revealed, for the first time, that PRDX3 may play important roles in neurite outgrowth and AD development., Competing Interests: Declaration of competing interest The authors declare that there was no potential conflict of interest existed., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Reelin is a secreted glycoprotein important for brain development and synaptic plasticity in the adult brain. Some reports suggest that Reelin is secreted from the nerve terminals and functions as a neurotransmitter. However, the mechanism of Reelin secretion is unknown. In this study, we visualized Reelin secretion by bioluminescence imaging using a fusion protein of Reelin and Gaussia luciferase (GLase-Reelin). GLase-Reelin expressed in HEK293T cells was correctly processed and secreted. Luminescence signals from the secreted GLase-Reelin of primary cultured neurons were visualized by bioluminescence microscopy. Reelin secretory events were observed at neurites and cell bodies. Bioluminescence imaging was also performed before and after KCl depolarization to compare the secretory events of Reelin and brain-derived neurotrophic factor (BDNF). The secretion of BDNF increased markedly shortly after depolarization. In contrast, the frequency of Reelin secretion did not change significantly by depolarization. Thus, Reelin secretion from neurites might not be regulated in a neuronal activity-dependent manner., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Parkinson's disease (PD) is neurodegenerative disorder with the pathological hallmarks of progressive degeneration of midbrain dopaminergic neurons from the substantia nigra (SN), and accumulation and spread of inclusions of aggregated α-synuclein (α-Syn). Since current PD therapies do not prevent neurodegeneration, there is a need to identify therapeutic targets that can prevent α-Syn-induced reductions in neuronal survival and neurite growth. We hypothesised that genes that are normally co-expressed with the α-Syn gene (SNCA), and whose co-expression pattern is lost in PD, may be important for protecting against α-Syn-induced dopaminergic degeneration, since broken correlations can be used as an index of functional misregulation. Gene co-expression analysis of the human SN showed that nuclear zinc finger HIT-type containing 1 (ZNHIT1) is co-expressed with SNCA and that this co-expression pattern is lost in PD. Overexpression of ZNHIT1 was found to increase deposition of the H2A.Z histone variant in SH-SY5Y cells, to promote neurite growth and to prevent α-Syn-induced reductions in neurite growth and cell viability. Analysis of ZNHIT1 co-expressed genes showed significant enrichment in genes associated with mitochondrial function. In agreement, bioenergetic state analysis of mitochondrial function revealed that ZNHIT1 increased cellular ATP synthesis. Furthermore, α-Syn-induced impairments in basal respiration, maximal respiration and spare respiratory capacity were not seen in ZNHIT1-overexpressing cells. These data show that ZNHIT1 can protect against α-Syn-induced degeneration and mitochondrial dysfunction, which rationalises further investigation of ZNHIT1 as a therapeutic target for PD., (© 2022. The Author(s).)

Numerous studies have reported neuroprotective and procognitive effects of estrogens. The estrogen 17β-estradiol (E2) activates both the classical nuclear estrogen receptors ERα and ERβ as well as the G protein-coupled estrogen receptor (GPER). The differential effects of targeting the classical estrogen receptors over GPER are not well-understood. A limited number of selective GPER compounds have been described. In this study, 10 novel compounds were synthesized and exhibited half-maximal effective concentration values greater than the known GPER agonist G-1 in calcium mobilization assays performed in nonadherent HL-60 cells. Of these compounds, 2-cyclohexyl-4-isopropyl- N -((5-(tetrahydro-2 H -pyran-2-yl)furan-2-yl)methyl)aniline, referred to as CITFA, significantly increased axonal and dendritic growth in neurons extracted from embryonic day 18 (E18) fetal rat hippocampal neurons. Confirmation of the results was performed by treating E18 hippocampal neurons with known GPER-selective antagonist G-36 and challenging with either E2, G-1, or CITFA. Results from these studies revealed an indistinguishable difference in neurite outgrowth between the treatment and control groups, exhibiting that neurite outgrowth in response to G-1 and CITFA originates from GPER activation and can be abolished with pretreatment of an antagonist. Subsequent docking studies using a homology model of GPER showed unique docking poses between G-1 and CIFTA. While docking poses differed between the ligands, CIFTA exhibited more favorable distance, bond angle, and strain for hydrogen-bonding and hydrophobic interactions.

Adhesion molecules regulate cell proliferation, migration, survival, neuritogenesis, synapse formation and synaptic plasticity during the nervous system's development and in the adult. Among such molecules, the neural cell adhesion molecule L1 contributes to these functions during development, and in synapse formation, synaptic plasticity and regeneration after trauma. Proteolytic cleavage of L1 by different proteases is essential for these functions. A proteolytic fragment of 70 kDa (abbreviated L1-70) comprising part of the extracellular domain and the transmembrane and intracellular domains was shown to interact with mitochondrial proteins and is suggested to be involved in mitochondrial functions. To further determine the role of L1-70 in mitochondria, we generated two lines of gene-edited mice expressing full-length L1, but no or only low levels of L1-70. We showed that in the absence of L1-70, mitochondria in cultured cerebellar neurons move more retrogradely and exhibit reduced mitochondrial membrane potential, impaired Complex I activity and lower ATP levels compared to wild-type littermates. Neither neuronal migration, neuronal survival nor neuritogenesis in these mutants were stimulated with a function-triggering L1 antibody or with small agonistic L1 mimetics. These results suggest that L1-70 is important for mitochondrial homeostasis and that its absence contributes to the L1 syndrome phenotypes.

During development and regeneration, growth cones at the tips of extending axons navigate through a complex environment to establish accurate connections with appropriate targets. Growth cones can respond rapidly to classical and non-classical guidance cues in their environment, often requiring local protein synthesis. In vertebrate growth cones, local protein synthesis in response to classical cues can require regulation by microRNAs (miRNAs), a class of small, conserved, non-coding RNAs that post-transcriptionally regulate gene expression. However, less is known of how miRNAs mediate growth cone responses to non-classical cues (such as retinoic acid (RA)), specifically in invertebrates. Here, we utilized adult regenerating invertebrate motorneurons to study miRNA regulation of growth cone attraction to RA, shown to require local protein synthesis. In situ hybridization revealed the presence of miR-124 in growth cones of regenerating ciliary motorneurons of the mollusc Lymnaea stagnalis. Changes in the spatiotemporal distribution of miR-124 occurred following application of RA, and dysregulation of miR-124 (with mimic injection), disrupted RA-induced growth cone turning in a time-dependent manner. This behavioural regulation by miR-124 was altered when the neurite was transected, and the growth cone completely separated from the soma. miR-124 did not, however, appear to be involved in growth cone attraction to serotonin, a response independent of local protein synthesis. Finally, we provide evidence that a downstream effector of RhoGTPases, ROCK, is a potential target of miR-124 during RA-induced growth cone responses. These data advance our current understanding of how microRNAs might mediate cue- and context-dependent behaviours during axon guidance., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Cell behaviour is influenced by external factors including the physical properties of the substrate such as its surface topography and stiffness. Recent studies have demonstrated the potential of aerogels as biomaterials and specifically as neural scaffolds. The 3-D structure inherent to aerogels offers an advantage over other biocompatible substrates which lack the dimensionality needed to mimic the in vivo topography of tissues. Here, we used a variety of aerogel types to correlate the extension of neurites by neuronal cells with surface roughness ranging from 0 to 3 μm and stiffness 10 kPa-4 MPa. This investigation reveals that the optimal surface features for neurite extension are a surface roughness of 0.5 μm and a Young's modulus between 1 and 3.5 MPa. The significance of these findings to optimize materials for nerve repair is discussed., (Copyright © 2022. Published by Elsevier B.V.)

The synthesis and bioactivity of neurotrophic banglenes and derivatives is described, establishing a structure-activity relationship which enables future mechanistic studies. Neuritogenesis assays indicate that (-) trans -banglene is the active enantiomer. Assays performed with and without NGF protein suggest that neurotrophic activity and potentiation of NGF activity by (-) trans -banglene might be distinct unassociated processes. Interestingly, (-) trans -banglene potentiation of NGF-induced neuritogenesis is unaffected by the presence of Erk1/2, Akt and Pkc inhibitors.

Neurodegenerative diseases are generally characterized clinically by the selective loss of a distinct subset of neurons and a slow progressive course. Mounting evidence in vivo indicates that large numbers of neurons pass through a long period of injury and dysfunction before the actual death of the cells. Whether these dying neurons can be rescued and return to a normal, functional state is uncertain. In the present study, we explored the reversibility of the neuronal cell death pathway at various stages by monitoring the dynamics of single cells with high-resolution live-cell spinning disk confocal microscopy in an in vitro neuronal cell death model. We exposed differentiated neuronal PC12 cells to ethanol as our cell death model. Results showed that exposure to 5% ethanol for 24 h induced cell death in >70% of the cells. Ethanol treatment for 3 h already induced cellular changes and damage such as reactive oxygen species generation, elevation of intracellular Ca2+ level, phosphatidylserine exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation and membrane potential loss, and retraction of neurites. These phenomena are often associated with programmed cell death. Importantly, after removing ethanol and further culturing these damaged cells in fresh culture medium, cells recovered from all these cell injuries and generated new neurites. Moreover, results indicated that this recovery was not dependent on exogenous NGF and other growth factors in the cell culture medium. Overall, our results suggest that targeting dying neurons can be an effective therapeutic strategy in neurodegenerative diseases.

Parkinson's disease (PD) is neuropathologically characterized by the loss of dopaminergic neurons and the deposition of aggregated alpha synuclein (aSyn). Mounting evidence suggests that neuritic degeneration precedes neuronal loss in PD. A possible underlying mechanism could be the interference of aSyn with microtubule organization in the neuritic development, as implied by several studies using cell-free model systems. In this study, we investigate the impact of aSyn on microtubule organization in aSyn overexpressing H4 neuroglioma cells and midbrain dopaminergic neuronal cells (mDANs) generated from PD patient-derived human induced pluripotent stem cells (hiPSCs) carrying an aSyn gene duplication ( SNCA Dupl ). An unbiased mass spectrometric analysis reveals a preferential binding of aggregated aSyn conformers to a number of microtubule elements. We confirm the interaction of aSyn with beta tubulin III in H4 and hiPSC-derived mDAN cell model systems, and demonstrate a remarkable redistribution of tubulin isoforms from the soluble to insoluble fraction, accompanied by a significantly increased insoluble aSyn level. Concordantly, SNCA Dupl mDANs show impaired neuritic phenotypes characterized by perturbations in neurite initiation and outgrowth. In summary, our findings suggest a mechanistic pathway, through which aSyn aggregation interferes with microtubule organization and induces neurite impairments.

Traumas and chronic damages can hamper the regenerative power of nervous, muscle, and connective tissues. Tissue engineering approaches are promising therapeutic tools, aiming to develop reliable, reproducible, and economically affordable synthetic scaffolds which could provide sufficient biomimetic cues to promote the desired cell behaviour without triggering graft rejection and transplant failure. Here, we used 3D-printing to develop 3D-printed scaffolds based on either PLA or graphene@PLA with a defined pattern. Multiple regeneration strategies require a specific orientation of implanted and recruited cells to perform their function correctly. We tested our scaffolds with induced pluripotent stem cells (iPSC), neuronal-like cells, immortalised fibroblasts and myoblasts. Our results demonstrated that the specific "lines and ridges" 100 µm-scaffold topography is sufficient to promote myoblast and fibroblast cell alignment and orient neurites along with the scaffolds line pattern. Conversely, graphene is critical to promote cells differentiation, as seen by the iPSC commitment to neuroectoderm, and myoblast fusions into multinuclear myotubes achieved by the 100 µm scaffolds containing graphene. This work shows the development of a reliable and economical 3D-printed scaffold with the potential of being used in multiple tissue engineering applications and elucidates how scaffold micro-topography and graphene properties synergistically control cell differentiation.

The extracellular matrix (ECM) plays a critical role in development, homeostasis, and regeneration of tissue structures and functions. Cell interactions with the ECM are dynamic and cells respond to ECM remodeling by changes in morphology and motility. During nerve regeneration, the ECM facilitates neurite outgrowth and guides axons with target specificity. Decellularized ECMs retain structural, biochemical, and biomechanical cues of native ECM and have the potential to replace damaged matrix to support cell activities during tissue repair. To determine the ECM components that contribute to nerve regeneration, we analyzed neuron-ECM interactions on two types of decellularized ECM. One matrix was composed primarily of fibronectin (FN) fibrils, and the other FN-rich ECM also contained significant numbers of type I collagen (COL I) fibrils. Using primary neurons dissociated from superior cervical ganglion (SCG) explants, we found that neurites were extended on both matrices without a significant difference in average neurite length after 24 h. The most distinctive features of neurites on the FN matrix were numerous short actin-filled protrusions and longer branches extending from neurite shafts. Very few protrusions and branches were detected on FN-COL matrix. Growth cone morphologies also differed with mostly filopodial growth cones on FN matrix whereas on FN-COL matrix, equivalent numbers of filopodial and slender growth cones were formed. Our work provides new information about how changes in major components of the ECM during tissue repair modulate neuron and growth cone morphologies and helps to define the contributions of neuron-ECM interactions to nerve development and regeneration., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aims: Although morphological attributes of cells and their substructures are recognised readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research., Methods: In this study, we integrate multichannel fluorescence high-content microscopy data with deep learning imaging methods to reveal-directly from unsegmented images-novel neurite-associated morphological perturbations associated with (ALS-causing) VCP-mutant human motor neurons (MNs)., Results: Surprisingly, we reveal that previously unrecognised disease-relevant information is withheld in broadly used and often considered 'generic' biological markers of nuclei (DAPI) and neurons ( β III-tubulin). Additionally, we identify changes within the information content of ALS-related RNA binding protein (RBP) immunofluorescence imaging that is captured in VCP-mutant MN cultures. Furthermore, by analysing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS., Conclusions: Our study therefore reveals disease-relevant information contained in a range of both generic and more specific fluorescent markers and establishes the use of image-based deep learning methods for rapid, automated and unbiased identification of biological hypotheses., (© 2021 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Intracellular transport is pivotal for cell growth and survival. Malfunctions in this process have been associated with devastating neurodegenerative diseases, highlighting the need for a deeper understanding of the mechanisms involved. Here, we use an experimental methodology that leads neurites of differentiated PC12 cells into either one of two configurations: a one-dimensional configuration, where the neurites align along lines, or a two-dimensional configuration, where the neurites adopt a random orientation and shape on a flat substrate. We subsequently monitored the motion of functional organelles, the lysosomes, inside the neurites. Implementing a time-resolved analysis of the mean-squared displacement, we quantitatively characterized distinct motion modes of the lysosomes. Our results indicate that neurite alignment gives rise to faster diffusive and super-diffusive lysosomal motion than the situation in which the neurites are randomly oriented. After inducing lysosome swelling through an osmotic challenge by sucrose, we confirmed the predicted slowdown in diffusive mobility. Surprisingly, we found that the swelling-induced mobility change affected each of the (sub-/super-)diffusive motion modes differently and depended on the alignment configuration of the neurites. Our findings imply that intracellular transport is significantly and robustly dependent on cell morphology, which might in part be controlled by the extracellular matrix.

The adapter protein SH2B1 is recruited to neurotrophin receptors, including TrkB (also known as NTRK2), the receptor for brain-derived neurotrophic factor (BDNF). Herein, we demonstrate that the four alternatively spliced isoforms of SH2B1 (SH2B1α-SH2B1δ) are important determinants of neuronal architecture and neurotrophin-induced gene expression. Primary hippocampal neurons from Sh2b1-/- [knockout (KO)] mice exhibit decreased neurite complexity and length, and BDNF-induced expression of the synapse-related immediate early genes Egr1 and Arc. Reintroduction of each SH2B1 isoform into KO neurons increases neurite complexity; the brain-specific δ isoform also increases total neurite length. Human obesity-associated variants, when expressed in SH2B1δ, alter neurite complexity, suggesting that a decrease or increase in neurite branching may have deleterious effects that contribute to the severe childhood obesity and neurobehavioral abnormalities associated with these variants. Surprisingly, in contrast to SH2B1α, SH2B1β and SH2B1γ, which localize primarily in the cytoplasm and plasma membrane, SH2B1δ resides primarily in nucleoli. Some SH2B1δ is also present in the plasma membrane and nucleus. Nucleolar localization, driven by two highly basic regions unique to SH2B1δ, is required for SH2B1δ to maximally increase neurite complexity and BDNF-induced expression of Egr1, Arc and FosL1., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The polarized flow of information through neural circuits depends on the orderly arrangement of neurons, their processes, and their synapses. This polarity emerges sequentially in development, starting with the directed migration of neuronal precursors, which subsequently elaborate neurites that form synapses in specific locations. In other organs, Fat cadherins sense the position and then polarize individual cells by inducing localized changes in the cytoskeleton that are coordinated across the tissue. Here, we show that the Fat-related protein Fat3 plays an analogous role during the assembly of polarized circuits in the murine retina. We find that the Fat3 intracellular domain (ICD) binds to cytoskeletal regulators and synaptic proteins, with discrete motifs required for amacrine cell migration and neurite retraction. Moreover, upon ICD deletion, extra neurites form but do not make ectopic synapses, suggesting that Fat3 independently regulates synapse localization. Thus, Fat3 serves as a molecular node to coordinate asymmetric cell behaviors across development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Auditory neurons connect the sensory hair cells from the inner ear to the brainstem. These bipolar neurons are relevant targets for pharmacological intervention aiming at protecting or improving the hearing function in various forms of sensorineural hearing loss. In the research laboratory, neurotrophic compounds are commonly used to improve survival and to promote regeneration of auditory neurons. One important roadblock delaying eventual clinical applications of these strategies in humans is the lack of powerful in vitro models allowing high throughput screening of otoprotective and regenerative compounds. The recently discovered auditory neuroprogenitors (ANPGs) derived from the A/J mouse with an unprecedented capacity to self-renew and to provide mature auditory neurons offer the possibility to overcome this bottleneck. In the present study, we further characterized the new phoenix ANPGs model and compared it to the current gold-standard spiral ganglion organotypic explant (SGE) model to assay neurite outgrowth, neurite length and glutamate-induced Ca 2+ response in response to neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF) treatment. Whereas both, SGEs and phoenix ANPGs exhibited a robust and sensitive response to neurotrophins, the phoenix ANPGs offer a considerable range of advantages including high throughput suitability, lower experimental variability, single cell resolution and an important reduction of animal numbers. The phoenix ANPGs in vitro model therefore provides a robust high-throughput platform to screen for otoprotective and regenerative neurotrophic compounds in line with 3R principles and is of interest for the field of auditory neuroscience., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (β-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1 . Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

During neurodevelopment, neurons form growth cones, F-actin rich extensions located at the distal end of the neurites. Growth cones allow dendrites and axons to build synaptic connections through a process of neurite guidance whose mechanisms have not been fully elucidated. Calcium is an important element in this process by inducing F-actin reorganization. We hypothesized that other biologically active elements might be involved in the growth cone-mediated neurite guidance mechanisms. We performed super resolution and confocal microscopy of F-actin, followed by synchrotron X-ray fluorescence microscopy of phosphorous, sulfur, chlorine, potassium, calcium, iron and zinc on growth cones from primary rat hippocampal neurons. We identified two main patterns of element organization. First, active growth cones presenting an asymmetric distribution of Ca co-localized with the cytoskeleton protein F-actin. In active growth cones, we found that the distributions of P, S, Cl, K, and Zn are correlated with Ca. This correlation is lost in the second pattern, quiescent growth cones, exhibiting a spread elemental distribution. These results suggest that Ca is not the only element required in the F-actin rich active regions of growth cones. In addition, highly concentrated Fe spots of submicrometer size were observed in calcium-rich areas of active growth cones. These results reveal the need for biological active elements in growth cones during neural development and may help explain why early life deficiencies of elements, such as Fe or Zn, induce learning and memory deficits in children., (© The Author(s) 2021. Published by Oxford University Press.)

Cell adhesion molecule L1 regulates multiple cell functions and L1 deficiency is linked to several neural diseases. Proteolytic processing generates functionally decisive L1 fragments, which are imported into the nucleus. By computational analysis, we found at L1's C-terminal end the chromo shadow domain-binding motif PxVxL, which directs the binding of nuclear proteins to the heterochromatin protein 1 (HP1) isoforms α, β, and ɣ. By enzyme-linked immunosorbent assay, we show that the intracellular L1 domain binds to all HP1 isoforms. These interactions involve the HP1 chromo shadow domain and are mediated via the sequence 1158 KDET 1161 in the intracellular domain of murine L1, but not by L1's C-terminal PxVxL motif. Immunoprecipitation using nuclear extracts from the brain and from cultured cerebellar and cortical neurons indicates that HP1 isoforms interact with a yet unknown nuclear L1 fragment of approximately 55 kDa (L1-55), which carries ubiquitin residues. Proximity ligation indicates a close association between L1-55 and the HP1 isoforms in neuronal nuclei. This association is reduced after the treatment of neurons with inhibitors of metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1), or ɣ-secretase, suggesting that cleavage of full-length L1 by these proteases generates L1-55. Reduction of HP1α, -β, or -ɣ expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons and decreases the L1-dependent migration of L1-transfected HEK293 cells in a scratch assay. These findings indicate that the interaction of the novel fragment L1-55 with HP1 isoforms in nuclei affects L1-dependent functions, such as neurite outgrowth and neuronal migration., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Recent studies have found that people with diabetes are more vulnerable to cognitive dysfunction, particularly Alzheimer's disease (AD). Previous studies revealed that Reticulon 3 (RTN3) oligomers could induce cortical neuritic dystrophy (CND) in the brains of diabetic rats. However, it is not clear how diabetes induces RTN3 aggregation. In this study, we examined in vivo and in vitro diabetes models to explore the underlying effects of RTN3-mediated neurite dystrophy. The results showed that the binding ability of ASC and RTN3 was significantly increased during diabetes- or high glucose-induced neuritic dystrophy, and ASC siRNA or an anti-inflammatory drug (CP 424174) could inhibit neuritic dystrophy in vitro. These results suggest that the ASC and RTN3 interaction is involved in diabetes-induced CND, and anti-inflammatory therapy might be an effective way to prevent and inhibit diabetes-induced CND., (Copyright © 2021 Elsevier B.V. All rights reserved.)

SP16 is an innovative peptide derived from the carboxyl-terminus of α1-Antitrypsin (AAT), corresponding to residues 364-380, and contains recognition sequences for the low-density lipoprotein receptor-related protein-1 (LRP1). LRP1 is an endocytic and cell-signaling receptor that regulates inflammation. Deletion of Lrp1 in Schwann cells increases neuropathic pain; however, the role of LRP1 activation in nociceptive and neuropathic pain regulation remains unknown. Herein, we show that SP16 is bioactive in sensory neurons in vitro. Neurite length and regenerative gene expression were increased by SP16. In PC12 cells, SP16 activated Akt and ERK1/2 cell-signaling in an LRP1-dependent manner. When formalin was injected into mouse hind paws, to model inflammatory pain, SP16 dose-dependently attenuated nociceptive pain behaviors in the early and late phases. In a second model of acute pain using capsaicin, SP16 significantly reduced paw licking in both male and female mice (p < .01) similarly to enzymatically inactive tissue plasminogen activator, a known LRP1 interactor. SP16 also prevented development of tactile allodynia after partial nerve ligation and this response was sustained for nine days (p < .01). Immunoblot analysis of the injured nerve revealed decreased CD11b (p < .01) and Toll-like receptor-4 (p < .005). In injured dorsal root ganglia SP16 reduced CD11b + cells (p < .05) and GFAP (p < .005), indicating that inflammatory cell recruitment and satellite cell activation were inhibited. In conclusion, administration of SP16 blocked pain-related responses in three distinct pain models, suggesting efficacy against acute nociceptive, inflammatory, and neuropathic pain. SP16 also attenuated innate immunity in the PNS. These studies identify SP16 as a potentially effective treatment for pain., (© 2021 Federation of American Societies for Experimental Biology.)

Mammalian TRPC5 channels are predominantly expressed in the brain, where they increase intracellular Ca 2+ and induce depolarization. Because they augment presynaptic vesicle release, cause persistent neural activity, and show constitutive activity, TRPC5s could play a functional role in late developmental brain events. We used immunohistochemistry to examine TRPC5 in the chick embryo brain between 8 and 20 days of incubation, and provide the first detailed description of their distribution in birds and in the whole brain of any animal species. Stained areas substantially increased between E8 and E16, and staining intensity in many areas peaked at E16, a time when chick brains first show organized patterns of whole-brain metabolic activation like what is seen consistently after hatching. Areas showing cell soma staining match areas showing Trpc5 mRNA or protein in adult rodents (cerebral cortex, hippocampus, amygdala, cerebellar Purkinje cells). Chick embryos show protein staining in the optic tectum, cerebellar nuclei, and several brainstem nuclei; equivalent areas in the Allen Institute mouse maps express Trpc5 mRNA. The strongest cell soma staining was found in a dorsal hypothalamic area (matching a group of parvicellular arginine vasotocin neurons and a pallial amygdalohypothalamic cell corridor) and the vagal motor complex. Purkinje cells showed strong dendritic staining at E20. Unexpectedly, we also describe neurite staining in the septum, several hypothalamic nuclei, and a paramedian raphe area; the strongest neurite staining was in the median eminence. These novel localizations suggest new unexplored TRPC5 functions, and possible roles in late embryonic brain development., (© 2021 Wiley Periodicals LLC.)

During neuronal development, immature neurons extend neurites and subsequently polarize to form an axon and dendrites. We have previously reported that G protein-coupled receptor 3 (GPR3) levels increase during neuronal development, and that GPR3 has functions in neurite outgrowth and neuronal differentiation in cerebellar granular neurons. Moreover, GPR3 is transported and concentrated at the tips of neurite, thereby contributing to the local activation of protein kinase A (PKA). However, the signaling pathways for GPR3-mediated neurite outgrowth and its subsequent effects on neuronal polarization have not yet been elucidated. We therefore analyzed the signaling pathways related to GPR3-mediated neurite outgrowth, and also focused on the possible roles of GPR3 in axon polarization. We demonstrated that, in cerebellar granular neurons, GPR3-mediated neurite outgrowth was mediated by multiple signaling pathways, including those of PKA, extracellular signal-regulated kinases (ERKs), and most strongly phosphatidylinositol 3-kinase (PI3K). In addition, the GPR3-mediated activation of neurite outgrowth was associated with G protein-coupled receptor kinase 2 (GRK2)-mediated signaling and phosphorylation of the C-terminus serine/threonine residues of GPR3, which affected downstream protein kinase B (Akt) signaling. We further demonstrated that GPR3 was transiently increased early in the development of rodent hippocampal neurons. It was subsequently concentrated at the tip of the longest neurite, and was thus associated with accelerated polarity formation in a PI3K-dependent manner in rat hippocampal neurons. In addition, GPR3 knockout in mouse hippocampal neurons led to delayed neuronal polarity formation, thereby affecting the dephosphorylation of collapsing response mediator protein 2 (CRMP2), which is downstream of the PI3K signaling pathway. Taken together, these findings suggest that the intrinsic expression of GPR3 in differentiated neurons constitutively activates PI3K-mediated signaling pathway predominantly, thus accelerating neurite outgrowth and further augmenting polarity formation in primary cultured neurons., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Diacylglycerol kinase β (DGKβ) is an enzyme that converts diacylglycerol to phosphatidic acid and is mainly expressed in the cerebral cortex, hippocampus and striatum. We previously reported that DGKβ induces neurite outgrowth and spinogenesis, contributing to higher brain functions, including emotion and memory. To elucidate the mechanisms involved in neuronal development by DGKβ, we investigated the importance of DGKβ activity in the induction of neurite outgrowth using human neuroblastoma SH-SY5Y cells. Interestingly, both wild-type DGKβ and the kinase-negative (KN) mutant partially induced neurite outgrowth, and these functions shared a common pathway via the activation of mammalian target of rapamycin complex 1 (mTORC1). In addition, we found that DGKβ interacted with the small GTPase RalA and that siRNA against RalA and phospholipase D (PLD) inhibitor treatments abolished DGKβKN-induced neurite outgrowth. These results indicate that binding of RalA and activation of PLD and mTORC1 are involved in DGKβKN-induced neurite outgrowth. Taken together with our previous reports, mTORC1 is a key molecule in both kinase-dependent and kinase-independent pathways of DGKβ-mediated neurite outgrowth, which is important for higher brain functions.

Loss-of-function mutations in the human vacuolar protein sorting the 13 homolog A (VPS13A) gene cause Chorea-acanthocytosis (ChAc), with selective degeneration of the striatum as the main neuropathologic feature. Very little is known about the VPS13A expression in the brain. The main objective of this work was to assess, for the first time, the spatiotemporal distribution of VPS13A in the mouse brain. We found VPS13A expression present in neurons already in the embryonic stage, with stable levels until adulthood. VPS13A mRNA and protein distributions were similar in the adult mouse brain. We found a widespread VPS13A distribution, with the strongest expression profiles in the pons, hippocampus, and cerebellum. Interestingly, expression was weak in the basal ganglia. VPS13A staining was positive in glutamatergic, GABAergic, and cholinergic neurons, but rarely in glial cells. At the cellular level, VPS13A was mainly located in the soma and neurites, co-localizing with both the endoplasmic reticulum and mitochondria. However, it was not enriched in dendritic spines or the synaptosomal fraction of cortical neurons. In vivo pharmacological modulation of the glutamatergic, dopaminergic or cholinergic systems did not modulate VPS13A concentration in the hippocampus, cerebral cortex, or striatum. These results indicate that VPS13A has remarkable stability in neuronal cells. Understanding the distinct expression pattern of VPS13A can provide relevant information to unravel pathophysiological hallmarks of ChAc.

Exogenous electrical fields have been explored in regenerative medicine to increase cellular expression of pro-regenerative growth factors. Adipose-derived stem cells (ASCs) are attractive for regenerative applications, specifically for neural repair. Little is known about the relationship between low-level electrical stimulation (ES) and ASC regenerative potentiation. In this work, patterns of ASC expression and secretion of growth factors (i.e., secretome) were explored across a range of ES parameters. ASCs were stimulated with low-level stimulation (20 mV/mm) at varied pulse frequencies, durations, and with alternating versus direct current. Frequency and duration had the most significant effects on growth factor expression. While a range of stimulation frequencies (1, 20, 1000 Hz) applied intermittently (1 h × 3 days) induced upregulation of general wound healing factors, neural-specific factors were only increased at 1 Hz. Moreover, the most optimal expression of neural growth factors was achieved when ASCs were exposed to 1 Hz pulses continuously for 24 h. In evaluation of secretome, apparent inconsistencies were observed across biological replications. Nonetheless, ASC secretome (from 1 Hz, 24 h ES) caused significant increase in neurite extension compared to non-stimulated control. Overall, ASCs are sensitive to ES parameters at low field strengths, notably pulse frequency and stimulation duration., (© 2021. Biomedical Engineering Society.)

The molecular mechanisms underlying morphological diversity in retinal cell types are poorly understood. We have previously reported that several members of the Copine family of Ca-dependent membrane adaptors are expressed in Retinal Ganglion Cells and transcriptionally regulated by Brn3 transcription factors. Several Copines are enriched in the retina and their over-expression leads to morphological changes -formation of elongated processes-, reminiscent of neurites, in HEK293 cells. However, the role of Copines in the retina is largely unknown. We now investigate Cpne4, a Copine whose expression is restricted to Retinal Ganglion Cells. Over-expression of Cpne4 in RGCs in vivo led to formation of large varicosities on the dendrites but did not otherwise visibly affect dendrite or axon formation. Protein interactions studies using yeast two hybrid analysis from whole retina cDNA revealed two Cpne4 interacting proteins-Host Cell Factor 1 and Morn2. Mass Spectrometry analysis of retina lysate pulled down using Cpne4 or its vonWillebrand A domain showed 207 interacting proteins. A Gene Ontology analysis of the discovered proteins suggests that Cpne4 is involved in several metabolic and signaling pathways in the retina., Competing Interests: The authors have declared that no competing interests exist.

This protocol provides an improved pipeline for dissociating intact projection neurons from adult mouse cortex for applications including droplet and plate-based single-cell RNA sequencing, qPCR, immunocytochemistry, and long-term in vitro cell culture. This protocol provides a robust and reproducible dissociation pipeline that uses exclusively off-the-shelf reagents, not requiring the use of expensive dissociation kits. The unique incubation steps, in combination with the FACS gating strategy, results in unparalleled enrichment for intact cortical neurons from the adult brain. For complete details on the use and execution of this protocol, please refer to Golan et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

BZD9L1 was previously described as a SIRT1/2 inhibitor with anti-cancer activities in colorectal cancer (CRC), either as a standalone chemotherapy or in combination with 5-fluorouracil. BZD9L1 was reported to induce apoptosis in CRC cells; however, the network of intracellular pathways and crosstalk between molecular players mediated by BZD9L1 is not fully understood. This study aimed to uncover the mechanisms involved in BZD9L1-mediated cytotoxicity based on previous and new findings for the prediction and identification of related pathways and key molecular players. BZD9L1-regulated candidate targets (RCTs) were identified using a range of molecular, cell-based and biochemical techniques on the HCT 116 cell line. BZD9L1 regulated major cancer pathways including Notch, p53, cell cycle, NFκB, Myc/MAX, and MAPK/ERK signalling pathways. BZD9L1 also induced reactive oxygen species (ROS), regulated apoptosis-related proteins, and altered cell polarity and adhesion profiles. In silico analyses revealed that most RCTs were interconnected, and were involved in the modulation of catalytic activity, metabolism and transcription regulation, response to cytokines, and apoptosis signalling pathways. These RCTs were implicated in p53-dependent apoptosis pathway. This study provides the first assessment of possible associations of molecular players underlying the cytotoxic activity of BZD9L1, and establishes the links between RCTs and apoptosis through the p53 pathway., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The aim of this study was to investigate the combined effect of mesenchymal stem cells (MSC) and local delivery of tacrolimus (FK506) on nerve regeneration when applied to nerve autografts and decellularized allografts., Methods: A three-dimensional in vitro compartmented cell culture system consisting of a neonatal dorsal root ganglion adjacent to a nerve graft was used to evaluate the regenerating neurites into the peripheral nerve scaffold. Nerve autografts and allografts were treated with (i) undifferentiated MSCs, (ii) FK506 (100 ng/mL) or (iii) both (N = 9/group). After 48 hours, neurite extension was measured to quantify nerve regeneration and stem cell viability was evaluated., Results: Stem cell viability was confirmed in all MSC-treated grafts. Neurite extension was superior in autografts treated with FK506, and MSCs and FK506 combined (p < 0.001 and p = 0.0001, respectively), and autografts treated with MSCs (p = 0.12) were comparable to untreated autografts. In allografts, FK506 treatment and combined treatment were superior to controls (p < 0.001 and p = 0.0001, respectively), and treatment with MSCs (p = 0.09) was comparable to controls. All autograft groups were superior compared to their respective allograft treatment group (p < 0.05) in neurite extension., Conclusions: Alone, either MSC or FK506 treatment improved neurite outgrowth, and combined they further enhanced neurite extension in both autografts and allografts., (© 2021 Wiley Periodicals LLC.)

Surgical intervention with the use of autografts is considered the gold standard to treat peripheral nerve injuries. However, a biomaterial that supports and guides nerve growth would be an attractive alternative to overcome problems with limited availability, morbidity at the site of harvest, and nerve mismatches related to autografts. Native spider silk is a promising material for construction of nerve guidance conduit (NGC), as it enables regeneration of cm-long nerve injuries in sheep, but regulatory requirements for medical devices demand synthetic materials. Here, we use a recombinant spider silk protein (NT2RepCT) and a functionalized variant carrying a peptide derived from vitronectin (VN-NT2RepCT) as substrates for nerve growth support and neurite extension, using a dorsal root ganglion cell line, ND7/23. Two-dimensional coatings were benchmarked against poly-d-lysine and recombinant laminins. Both spider silk coatings performed as the control substrates with regards to proliferation, survival, and neurite growth. Furthermore, NT2RepCT and VN-NT2RepCT spun into continuous fibers in a biomimetic spinning set-up support cell survival, neurite growth, and guidance to an even larger extent than native spider silk. Thus, artificial spider silk is a promising biomaterial for development of NGCs., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The sigma 1 receptor (S1R) is an enigmatic ligand-operated chaperone involved in many important biological processes, and its functions are not fully understood yet. Herein, we developed a novel series of bitopic S1R ligands as versatile tools to investigate binding processes, allosteric modulation, and the oligomerization mechanism. These molecules have been prepared in the enantiopure form and subjected to a preliminary biological evaluation, while in silico investigations helped to rationalize the results. Compound 7 emerged as the first bitopic S1R ligand endowed with low nanomolar affinity ( K i = 2.6 nM) reported thus far. Computational analyses suggested that 7 may stabilize the open conformation of the S1R by simultaneously binding the occluded primary binding site and a peripheral site on the cytosol-exposed surface. These findings pave the way to new S1R ligands with enhanced activity and/or selectivity, which could also be used as probes for the identification of a potential allosteric site.