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The mammalian inner ear mediates hearing and balance and during development generates both cochleo-vestibular ganglion neurons and sensory epithelial receptor cells, that is, hair cells and support cells. Cell marking experiments have shown that both hair cells and support cells can originate from a common progenitor. Here, we demonstrate the lineage potential of individual otic epithelial cell clones using three cell lines established by a combination of limiting dilution and gene-marking techniques from an embryonic day 12 (E12) rat otocyst. Cell-type specific marker analyses of these clonal lines under proliferation and differentiation culture conditions demonstrate that during differentiation immature cell markers (Nanog and Nestin) were downregulated and hair cell (Myosin VIIa and Math1), support cell (p27 Kip1 and cytokeratin) and neuronal cell (NF-H and NeuroD) markers were upregulated. Our results suggest that the otic epithelium of the E12 mammalian inner ear possess multipotent progenitor cells able to generate cell types of both sensory epithelial and neural cell lineages when cultured under a differentiation culture condition. Understanding the molecular mechanisms of proliferation and differentiation of multipotent otic progenitor cells may provide insights that could contribute to the development of a novel cell therapy with a potential to initiate or stimulate the sensorineural repair of damaged inner ear sensory receptors. Anat Rec, 303:451-460, 2020. © 2019 American Association for Anatomy., (© 2020 American Association for Anatomy.)

In addition to its role in the treatment of pancreatitis, the serine protease inhibitor nafamostat exhibits a retinal protective effect. However, the exact mechanisms underlying this effect are unknown. In this study, the neuroprotective effects of nafamostat and its orally active derivative sepimostat against excitotoxicity were further characterised in vitro and in vivo. In primary rat cortical neurons, nafamostat completely suppressed N-methyl-D-aspartate (NMDA)-induced cell death. Intravitreal injection of nafamostat and sepimostat protected the rat retina against NMDA-induced degeneration, whereas the structurally related compounds, gabexate and camostat, did not. The neuroprotective effects of nafamostat and the NR2B antagonist ifenprodil were remarkably suppressed by spermidine, a naturally occurring polyamine that modulates the NR2B subunit. Both nafamostat and sepimostat inhibited [ 3 H]ifenprodil binding to fractionated rat brain membranes. Thus, nafamostat and sepimostat may exert neuroprotective effects against excitotoxic retinal degeneration through NMDA receptor antagonism at the ifenprodil-binding site of the NR2B subunit.

Recently, we showed that imidazole dipeptide such as carnosine contained abundantly in chicken breast meat improves brain function in a double-blind randomized controlled trial. However, the underlying molecular mechanisms remain unknown. Here, we investigated whether carnosine activates intestinal epithelial cells and induces the secretion of factors that activate brain function. We focused on exosomes derived from intestinal epithelial cells as mediators of brain-gut interaction. Results showed that exosomes derived from Caco-2 cells treated with carnosine significantly induced neurite growth in SH-SY5Y cells. To clarify the molecular basis of this finding, we performed integrated analysis of microRNAs (miRNAs) with altered expression in exosomes in response to carnosine treatment and mRNAs with altered expression in target cells in response to exosome treatment to identify related miRNAs and their target genes. The combination of miR-6769-5p and its target gene ATXN1 was found to be involved in the exosome-induced activation of neuronal cells., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: NH Foods Ltd provided funding for this study and employ the authors [MS, TH]. This does not alter our adherence to PLOS ONE policy on sharing data and materials.

The brain is composed of many different types of neurons. Therefore, analysis of brain activity with single-cell resolution could provide fundamental insights into brain mechanisms. However, the electrical signal of an individual neuron is very small, and precise isolation of single neuronal activity from moving subjects is still challenging. To measure single-unit signals in actively behaving states, establishment of technologies that enable fine control of electrode positioning and strict spike sorting is essential. To further apply such a single-cell recording approach to small brain areas in naturally behaving animals in large spaces or during social interaction, we developed a compact wireless recording system with a motorized microdrive. Wireless control of electrode placement facilitates the exploration of single neuronal activity without affecting animal behaviors. Because the system is equipped with a newly developed data-encoding program, the recorded data are readily compressed almost to theoretical limits and securely transmitted to a host computer. Brain activity can thereby be stably monitored in real time and further analyzed using online or offline spike sorting. Our wireless recording approach using a precision motorized microdrive will become a powerful tool for studying brain mechanisms underlying natural or social behaviors.

Short interspersed repetitive elements (SINEs) are highly repeated sequences that account for a significant proportion of many eukaryotic genomes and are usually considered "junk DNA". However, we previously discovered that many AmnSINE1 loci are evolutionarily conserved across mammalian genomes, suggesting that they may have acquired significant functions involved in controlling mammalian-specific traits. Notably, we identified the AS021 SINE locus, located 390 kbp upstream of Satb2. Using transgenic mice, we showed that this SINE displays specific enhancer activity in the developing cerebral cortex. The transcription factor Satb2 is expressed by cortical neurons extending axons through the corpus callosum and is a determinant of callosal versus subcortical projection. Mouse mutants reveal a crucial function for Sabt2 in corpus callosum formation. In this study, we compared the enhancer activity of the AS021 locus with Satb2 expression during telencephalic development in the mouse. First, we showed that the AS021 enhancer is specifically activated in early-born Satb2(+) neurons. Second, we demonstrated that the activity of the AS021 enhancer recapitulates the expression of Satb2 at later embryonic and postnatal stages in deep-layer but not superficial-layer neurons, suggesting the possibility that the expression of Satb2 in these two subpopulations of cortical neurons is under genetically distinct transcriptional control. Third, we showed that the AS021 enhancer is activated in neurons projecting through the corpus callosum, as described for Satb2(+) neurons. Notably, AS021 drives specific expression in axons crossing through the ventral (TAG1(-)/NPY(+)) portion of the corpus callosum, confirming that it is active in a subpopulation of callosal neurons. These data suggest that exaptation of the AS021 SINE locus might be involved in enhancement of Satb2 expression, leading to the establishment of interhemispheric communication via the corpus callosum, a eutherian-specific brain structure.

The mechanisms of intracellular calcium store depletion and store-related Ca(2+) dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca(2+) by BAPTA/AM (50 microM), or La(3+) (100 microM) enhanced while dantrolene (100 microM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca(2+) stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca(2+) concentrations ([Ca(2+)](i)).

To investigate the effect of estrogen on neuronal differentiation, especially on dopaminergic (DA) neurons, human neural stem cells (NSCs) were differentiated in the presence of 17beta-estradiol. NSCs gave rise to tyrosine hydroxylase (TH)-positive neurons in vitro, the proportion of which was increased by 17beta-estradiol. Increase in TH-positive neurons was abrogated by an estrogen receptor (ER) antagonist, ICI182780, suggesting ERs play a role in differentiation of DA neurons. The observation that ERs were expressed in both proliferating NSCs and postmitotic DA neurons suggested that increase in TH-positive neurons was due to induction and support of DA neurons. 17beta-Estradiol also increased the number of DA neurons derived from human NSCs in vivo when the cells were grafted into mouse brains. These results support a possible role for estrogen in the transplantation of NSCs for Parkinson's disease.

Recent studies have revealed that neural precursor cells can be expanded not only from the subventricular zone and hippocampus but also from other regions of the human embryonic brain. To determine the regional differences of these precursor cells, we divided the brain of a 9-week-old human embryo into four parts, i.e., telencephalon, diencephalon, mesencephalon, and rhombencephalon. All cultures of the tissues yielded neurospheres, and these spheres gave rise to neurons, astrocytes, and oligodendrocytes. An analysis of clonal populations revealed that these precursor cells were multipotent, and two region-specific differences in neural precursor cells were revealed: 1) The precursor cells from the rostral part of the brain tended to proliferate faster than those from the caudal part, and 2) the precursor cells from the diencephalon and mesencephalon gave rise to more tyrosine hydoxylase (TH)-positive neurons than those from the telencephalon and rhombencephalon. When 50-day-cultured spheres were caused to differentiate, the percentage of TH-positive cells per total cell population was 1.2% for diencephalic and mesencephalic precursors, whereas it was 0.4% for telencephalic and rhombencephalic ones. Furthermore, the TH-positive cells from diencephalic and mesencephalic precursors were large, multipolar, and gamma-aminobutyric acid (GABA)-negative, which suggested that these cells were midbrain dopaminergic neurons. In contrast, TH-positive cells from telencephalic and rhombencephalic precursors were small, bipolar, and GABA-positive. These results suggest that human neural precursor cells might have the potential to differentiate into a variety of cells but retain regional specificity., (Copyright 2004 Wiley-Liss, Inc.)

Glycation, one of the post-translational modifications of proteins, is a nonenzymatic reaction initiated by the primary addition of a sugar aldehyde or ketone to the amino groups of proteins. In the early stage of glycation, the synthesis of intermediates leading to the formation of Amadori compounds occurs. In the late stage, advanced glycation end products (AGE) are irreversibly formed after a complex cascade of reactions. Several AGEs have been characterized chemically, while other new compounds remain to be identified. To date, studies of the contribution of glycation to diseases have been primarily focused on its relationship to diabetes and diabetes-related complications. However, glucose-induced damage is not limited to diabetic patients. Although it does not cause rapid or remarkable cell damage, glycation advances slowly and accompanies every fundamental process of cellular metabolism. It has recently become clear that glycation also affects physiological aging and neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Glycation alters the biological activity of proteins and their degradation processes. Protein cross-linking by AGE results in the formation of detergent-insoluble and protease-resistant aggregates. Such aggregates may interfere with both axonal transport and intracellular protein traffic in neurons. In addition, glycation reactions lead to the production of reactive oxygen species. Conversely, glycation is promoted by oxidative stress. We speculate on the presence of synergism between glycation and oxidative stress. In this review, we provide an outline of glycation and propose some possible mechanisms of its cytotoxicity and defense systems against it.

Proteasomal dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD). We examined the effect of a selective proteasomal inhibitor, epoxomicin, on primary cultured mesencephalic neurons. Exposing rat cultured mesencephalic neurons to epoxomicin for 24 h resulted in neurotoxicity in a dose-dependent manner. Epoxomicin caused mitochondrial dysfunction, reduction in reduced glutathione (GSH), and increased generation of free radicals. Neuronal damage was significantly blocked by antioxidative/GSH-augmenting agents. Epoxomicin also increased the expression of Bax and decreased that of Bcl-2, which may cause mitochondrial dysfunction and release of free radicals. Dopaminergic neurons were preferentially resistant to the toxicity of epoxomicin. Inhibiting the synthesis of tetrahydrobiopterin (BH(4)), which has been reported to have antioxidative function, increased the susceptibility of dopaminergic neurons, whereas increasing BH(4) levels protected non-dopaminergic neurons. These findings suggest that BH(4) is at least in part a contributing factor to grand the resistance to dopaminergic neurons against epoxomicin neurotoxicity. Our results suggest that proteasome inhibition causes the neurotoxicity in mesencephalic neurons, but that is not sufficient to reproduce the selective damage to dopaminergic neurons, such as that seen in PD.

Brefeldin A (BFA) is a fungus metabolite that is known to cause the disassembly of the Golgi complex and apoptosis in exposed cells, both of which have been suggested as playing roles in the pathogenesis of neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS). This study showed that BFA caused neurotoxicity and apoptotic nuclear changes in cultured spinal neurons of rat spinal cord in a dose- and time-dependent manner. The spinal motor neurons were more vulnerable to this neurotoxicity. The cultured spinal neurons showed irreversible disassembly of the Golgi apparatus as early as 1 hr after exposure to BFA. BFA induced the expression and activation of caspase-12 beginning 8 hr after exposure. The level of the cleaved form of caspase-3 had increased 12 hr after the addition of BFA. Free radical generation and loss of mitochondrial membrane potential were observed in the later stages of neurotoxicity caused by BFA. Collectively, our data suggests that BFA is an excellent agent for reproducing the pathophysiological features of ALS. This in vitro model may be useful in attempts to study the mechanisms of this neurodegenerative disease and to examine therapeutic potentials., (Copyright 2002 Wiley-Liss, Inc.)

We investigated the effect of two proteasome inhibitors, lactacystin and epoxomicin, on cultured spinal cord neurons. The incubation of spinal neurons with proteasome inhibitors for 24 hr induced neurotoxicity in a dose-dependent manner. We found motor neurons to be more vulnerable to proteasome-induced neurotoxicity than nonmotor neurons. The staining of cell bodies in treated motor neurons was markedly disrupted and showed characteristic granular patterns. Proteasome-induced neurotoxicity is accompanied by apoptotic nuclear changes, posttranslational modification of the cellular proteins, generation of intracellular free radicals, reduction in the amount of reduced glutathione, and mitochondrial dysfunction. Neurotoxicity was reduced by the administration of low concentrations (1-100 nM) of geranylgeranylacetone (GGA), which is widely used as an antiulcer drug, although higher concentrations of this drug produced neurotoxicity in spinal cord neurons. GGA was found to induce the expression of heat shock protein 70 as well as thioredoxin, which may partly contribute to the protective effect of GGA. These data suggest that the inhibition of proteasome may play a role in the mechanism of neurodegenerative diseases of the spinal cord, such as amyotrophic lateral sclerosis, and that the use of GGA may be effective in the treatment of these conditions., (Copyright 2002 Wiley-Liss, Inc.)

The effects of prostaglandin (PG) E(1) on NO neurotoxicity were examined using rat cultured spinal neurons. Rat cultured spinal neurons exposed to the NO donor, 2,2'-(hydroxynitrosohydrazono) bis-ethanamine (NOC18), showed neurotoxic effects that were accompanied by apoptotic nuclear change, free radical generation, a reduction in glutathione, and mitochondrial dysfunction. PGE(1), at concentrations of 1-100 nM, protected cultured spinal neurons from NO toxicity by reversing the oxidative and pro-apoptotic properties elicited by NOC18 exposure. The administration of PGE(1) increased the intracellular cyclic AMP (cAMP) levels in cultured spinal neurons. In addition, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed the existence of EP4, a cAMP-elevating PGE receptor, in cultured spinal neurons. The protective effects of PGE(1) against NO neurotoxicity was partially blocked by an inhibitor of MEK [the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase], suggesting that the MAPK/ERK pathway may play a significant role in the activity of PGE(1). PGE(1) up-regulated the expression of the anti-apoptotic protein, Bcl-2, as determined by Western blot analysis. PGE(1) also induced the expression of thioredoxin in cultured spinal neurons. Our data indicate that PGE(1) exerts a protective action against NO neurotoxicity in cultured spinal neurons, and suggests a therapeutic potential of PGE(1) against spinal cord disease, such as amyotrophic lateral sclerosis.

A decrease in intracellular glutathione content may be related to the primary event in Parkinson's disease, so increasing the glutathione level may have a therapeutic benefit. The biologically active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1, 25-(OH)(2)D(3)] has been recently reported to enhance the intracellular glutathione concentration in the central nervous system. Exposing rat cultured mesencephalic neurons for 24 hr to a mixture of L-buthionine sulfoximine (BSO) and 1-methyl-4-phenylpyridium ions (MPP(+)) resulted in a relatively selective damage to dopaminergic neurons. This damage has been accompanied by a reduction of intracellular glutathione levels. Low doses, i.e., 1-100 nM, of 1,25-(OH)(2)D(3) protect cultured dopaminergic neurons against this toxicity, although higher concentrations of this active form of vitamin D have been found to enhance the toxic effect. Generation of reactive oxygen species (ROS) by this toxicity has been attenuated in cultures being pretreated with low concentrations of 1,25-(OH)(2)D(3). Because the hormone increases the intracellular glutathione content in cultures, determining how this hormone suppresses ROS generation may involve the enhancement of the antioxidative system. These data suggest that low doses of 1,25-(OH)(2)D(3) are able to protect mesencephalic dopaminergic neurons against BSO/MPP(+)-induced toxicity that causes a depletion in glutathione content., (Copyright 2000 Wiley-Liss, Inc.)

In this study, we investigate the neurotoxicity of glycation, particularly early-stage glycation, and its mechanisms, which are possibly synergized with oxidative stress. Methylglyoxal (MG) and 3-deoxyglucosone (3DG), intermediate products of glycation, are known to further accelerate glycation and advanced glycation endproducts (AGEs) formation. Both compounds showed neurotoxicity on cultured cortical neurons and these effects were associated with reactive oxygen species production followed by neuronal apoptosis. Pretreatment with N-acetylcysteine induced neuroprotection against MG and 3DG. Cotreatment, but not pretreatment, with aminoguanidine protected neurons against the neurotoxicities of both compounds. The present study provides the first evidence that MG and 3DG are neurotoxic to cortical neurons in culture. Interference with the process by which glycation and AGEs formation occur may provide new therapeutic opportunities to reduce the pathophysiological changes associated with neurodegeneration, if, as indicated here, the participation of glycoxidation in the pathogenesis of neurodegenerative diseases is essential., (Copyright 1999 Wiley-Liss, Inc.)

Pretreatments with TNF-alpha and lower concentrations of C2-ceramide protected cultured mesencephalic neurons from excitotoxicity in a dose-dependent manner. These protective effects are reduced by cotreatment with N,N-dimethylsphingosine (DMS), an inhibitor of sphingosine kinase. Since the pretreatment with sphingosine-1-phosphate (SPP) showed a neuroprotective effect, our data suggest that protective effects of TNF and C2-ceramide could be attributable to their further metabolism to SPP., (Copyright 1999 Elsevier Science B.V.)

The rat embryonic brain was probed with anti-d-aspartic acid (d-Asp) antiserum at different stages of development. At gestational day (E) 12, weak immunoreactivity (IR) of d-Asp was apparent at the hindbrain, midbrain and caudal forebrain, whereas it became more intense and extended over the whole brain at E20. However, IR markedly decreased after parturition. In the region of the immature forebrain at an early stage of development (E12), IR was mainly a characteristic of the cytoplasm of the neuronal cells, while in the more mature hindbrain it was localized in the axonal zone. In the more differentiated forebrain at a later stage of development (E18), the IR became restricted to zones which mainly consisted of axons and processes. Consequently, in the rat central nervous system, d-Asp first emerges during embryonic development as a feature of the cytoplasm and thereafter spreads into the axonal regions of neuronal cells, before disappearing almost completely after parturition., (Copyright 1998 Elsevier Science B.V.)

The effect of recombinant human tumor necrosis factor-alpha (TNF) on voltage-gated membrane currents of cultured neurons derived from embryonic rat cerebral cortex was studied using the whole-cell patch-clamp technique. Treatment of neurons with TNF resulted in an increase in outward potassium current density, dependent upon the concentration of TNF and the incubation time, without affecting other membrane currents such as barium and N-methyl-D-aspartate (NMDA). Long exposures (12-48 hr) to TNF (10-100 ng/ml) increased transient outward potassium current (A-current) density without affecting the parameters of activation and inactivation of the current. Prolonged exposures to TNF diminished its increasing effect on the A-current. Since the increase of A-current density induced by TNF is inhibited by both the anti-TNF receptor antibody and cycloheximide treatment, the effect of TNF might be mediated through receptors and by de novo synthesis of the channel protein itself and/or modulating proteins associated with the channel activities. Results indicate that phosphatidylcholine-specific phospholipase C and protein kinase C, but not ceramide, are involved in the signal transduction. In toxicological experiments, TNF had no neurotoxicity. Moreover, a 12 hr pretreatment of TNF protected neurons against NMDA-induced neurotoxicity. This protective effect of TNF was cancelled by 4-aminopyridine, an A-current blocker, suggesting that the increase of A-current densities induced by TNF contributes to the neuroprotection.

Laminin is a large basement membrane glycoprotein which influences the behavior and morphology of a variety of cells. We have found that laminin and a pepsin fragment of laminin (P-lam) contain distinct sites for HT-1080 human fibrosarcoma cell attachment and for neurite outgrowth activity of PC12 and NG108-15 cell lines. Reduction and alkylation of laminin and P-lam fragment disulfide bonds, in the absence of denaturing agents, markedly reduced the cell attachment activity without reducing the neurite outgrowth response. The P-lam fragment (approximately 375 kDa) was found to contain part of the cross region of laminin and a portion of the long arm, on the basis of recognition by antisera against laminin synthetic peptides and fusion proteins. Modification of arginine residues by cyclohexanedione also had no effect on neurite outgrowth but reduced HT-1080 cell adhesion. Modification of lysine residues by succinic and citraconic anhydride, however, abolished laminin neurite outgrowth but not cell attachment activity. Neurite outgrowth activity was recovered by reversing the lysine modification. These data support the existence on laminin of separate sites for cell attachment and for neurite outgrowth.

Cell bodies with vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) were found in the thalamus of the rat. They were distributed throughout the ventrolateral nucleus (VL) and in the whole extent of the thalamic reticular nucleus (R) except for its most rostral part. On the basis of soma diameters, VIP-LI cells in the VL and R were assumed to be projection neurons.

Neurons from peripheral and central nervous tissue as well as from established cell lines respond to low concentrations of laminin with rapid extension of axon-like processes. Two sites on laminin have been identified which stimulate neurite outgrowth, the major site residing at the end of the long arm of laminin. Recently laminin has been cloned and sequenced allowing for synthetic peptides to be prepared and tested for biological activity. We report here that antisera against synthetic peptides corresponding to A and to B1 chain sequences at the end of the long arm can partially inhibit laminin-mediated neurite outgrowth. Further, we show that a 19 amino acid synthetic peptide (CSRARKQAASIKVAVSADR) from the long arm of the laminin A chain is capable of stimulating neuronal-like process formation to almost the same extent as laminin and competes with laminin for stimulation of neurite outgrowth.