Ribosomal Protein Dynamics and Its Association with Actin Filaments and Local Translation in Axonal Growth Cones of Dorsal Root Ganglia Neurons.

Local translation in growth cones plays a critical role in responses to extracellular stimuli, such as axon guidance cues. We previously showed that brain-derived neurotrophic factor activates translation and enhances novel protein synthesis through the activation of mammalian target of rapamycin complex 1 signaling in growth cones of dorsal root ganglion neurons. In this study, we focused on 40S ribosomal protein S6 (RPS6), 60S ribosomal protein P0/1/2 (RPP0/1/2), and actin filaments to determine how localization of ribosomal proteins changes with overall protein synthesis induced by neurotrophins. Our quantitative analysis using immunocytochemistry and super-resolution microscopy indicated that RPS6, RPP0/1/2, and actin tend to colocalize in the absence of stimulation, and that these ribosomal proteins tend to dissociate from actin and associate with each other when local protein synthesis is enhanced. We propose that this is because stimulation causes ribosomal subunits to associate with each other to form actively translating ribosomes (polysomes). This study further clarifies the role of cytoskeletal components in local translation in growth cones.

Epidermal Growth Factor Suppresses the Development of GABAergic Neurons Via the Modulation of Perineuronal Net Formation in the Neocortex of Developing Rodent Brains.

Previously, we reported that epidermal growth factor (EGF) suppresses GABAergic neuronal development in the rodent cortex. Parvalbumin-positive GABAergic neurons (PV neurons) have a unique extracellular structure, perineuronal nets (PNNs). PNNs are formed during the development of PV neurons and are mainly formed from chondroitin sulfate (CS) proteoglycans (CSPGs). We examined the effect of EGF on CSPG production and PNN formation as a potential molecular mechanism for the inhibition of inhibiting GABAergic neuronal development by EGF. In EGF-overexpressing transgenic (EGF-Tg) mice, the number of PNN-positive PV neurons was decreased in the cortex compared with that in wild-type mice, as in our previous report. The amount of CS and neurocan was also lower in the cortex of EGF-Tg mice, with a similar decrease observed in EGF-treated cultured cortical neurons. PD153035, an EGF receptor (ErbB1) kinase inhibitor, prevented those mentioned above excess EGF-induced reduction in PNN. We explored the molecular mechanism underlying the effect of EGF on PNNs using fluorescent substrates for matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). EGF increased the enzyme activity of MMPs and ADAMs in cultured neurons. These enzyme activities were also increased in the EGF-Tg mice cortex. GM6001, a broad inhibitor of MMPs and ADAMs, also blocked EGF-induced PNN reductions. Therefore, EGF/EGF receptor signals may regulate PNN formation in the developing cortex.

The dual role of dopamine in the modulation of information processing in the prefrontal cortex underlying social behavior.

Dopamine in the prefrontal cortex is essential for the regulation of social behavior. However, stress-causing social withdrawal also promotes dopamine release in the prefrontal cortex. Thus, this evidence suggests opposite functions of dopamine in the prefrontal cortex. However, the influence of dopamine on prefrontal functions is yet to be fully understood. Here, we show that dopamine differentially modulated the neuronal activity triggered by social stimuli in the prefrontal cortex, depending on the duration of the dopamine activation (transient or sustained activation). Using chemogenetic techniques, we have found that social behavior was negatively regulated by a sustained increase in dopamine neuronal activity in the ventral tegmental area, while it was positively regulated by an acute increase. The duration of social interactions was positively correlated with the transient dopamine release triggered by social stimuli in the prefrontal cortex and negatively correlated with the sustained increase in prefrontal dopamine levels. Furthermore, the elevation of neural calcium signal, triggered by social stimuli, in the prefrontal cortex was attenuated by the persistent elevation of prefrontal dopamine levels, whereas an acute increase in dopamine levels enhanced it. Additionally, the chronic excess of dopamine suppressed c-Fos induction triggered by social stimuli in prefrontal neurons expressing dopamine D1 receptors, but not D2 receptors. These results suggest that sustained activation of prefrontal dopamine, at the opposite of its transient activation, can reduce prefrontal activity associated with social behavior, even for identical dopamine concentrations. Thus, dopamine plays opposite roles in modulating prefrontal activity depending on the duration of its action.

Multi-Harmonic Modulation in a Fiber-Optic Gyroscope.

Optimizing the bias modulation of a fiber-optic gyroscope is crucial to improving its precision. In this study, we propose and demonstrate the use of multiple harmonics of sinusoidal modulation as an intermediate alternative to the widely used modulation methods: sinusoidal and square-wave modulation. We show that this alternative integrates the advantages of each modulation method by providing a smooth modulation that produces a clean, spike-free output and a satisfactory signal-to-noise ratio. By using three harmonics of modulation in combination with a high frequency to reduce thermal phase noise, we obtained an angular random walk of 5.2(2)µdeg/h and a bias instability of ∼10µdeg/h. This is the highest performance ever reported for fiber-optic gyroscopes.

EGF Downregulates Presynaptic Maturation and Suppresses Synapse Formation In Vitro and In Vivo.

Neuronal differentiation, maturation, and synapse formation are regulated by various growth factors. Here we show that epidermal growth factor (EGF) negatively regulates presynaptic maturation and synapse formation. In cortical neurons, EGF maintained axon elongation and reduced the sizes of growth cones in culture. Furthermore, EGF decreased the levels of presynaptic molecules and number of presynaptic puncta, suggesting that EGF inhibits neuronal maturation. The reduction of synaptic sites is confirmed by the decreased frequencies of miniature EPSCs. In vivo analysis revealed that while peripherally administrated EGF decreased the levels of presynaptic molecules and numbers of synaptophysin-positive puncta in the prefrontal cortices of neonatal rats, EGF receptor inhibitors upregulated these indexes, suggesting that endogenous EGF receptor ligands suppress presynaptic maturation. Electron microscopy further revealed that EGF decreased the numbers, but not the sizes, of synaptic structures in vivo. These findings suggest that endogenous EGF and/or other EGF receptor ligands negatively modulates presynaptic maturation and synapse formation.

Familial idiopathic basal ganglia calcification with a heterozygous missense variant (c.902C>T/p.P307L) in SLC20A2 showing widespread cerebrovascular lesions.

We describe a postmortem case of familial idiopathic basal ganglia calcification (FIBGC) in a 72-year-old Japanese man. The patient showed progressive cognitive impairment with a seven-year clinical course and calcification of the basal ganglia, thalami, and cerebellar dentate nuclei. A novel heterozygous missense variant in SLC20A2 (c.920C>T/p.P307L), a type III sodium-dependent phosphate transporter (PiT-2), was subsequently identified, in addition to typical neuropathological findings of FIBGC, such as capillary calcification of the occipital gray matter, confluent calcification of the basal ganglia and cerebellar white matter, widespread occurrence of vasculopathic changes, cerebrovascular lesions, and vascular smooth muscle cell depletion. Immunohistochemistry for PiT-2 protein revealed no apparent staining in endothelial cells in the basal ganglia and insular cortex; however, the immunoreactivity in endothelial cells of the cerebellum was preserved. Moreover, Western blot analysis identified preserved PiT-2 immunoreactivity signals in the frontal cortex and cerebellum. The variant identified in the present patient could be associated with development of FIBGC and is known to be located at the large intracytoplasmic part of the PiT-2 protein, which has potential phosphorylation sites with importance in the regulation of inorganic phosphate transport activity. The present case is an important example to prove that FIGBC could stem from a missense variant in the large intracytoplasmic loop of the PiT-2 protein. Abnormal clearance of inorganic phosphate in the brain could be related to the development of vascular smooth muscle damage, the formation of cerebrovascular lesions, and subsequent brain calcification in patients with FIBGC with SLC20A2 variants.

Ultrafast Coherent Control of Condensed Matter with Attosecond Precision.

Coherent control is a technique to manipulate wave functions of matter with light. Coherent control of isolated atoms and molecules in the gas phase is well-understood and developed since the 1990s, whereas its application to condensed matter is more difficult because its coherence lifetime is shorter. We have recently applied this technique to condensed matter samples, one of which is solid para-hydrogen ( p-H2). Intramolecular vibrational excitation of solid p-H2 gives an excited vibrational wave function called a "vibron", which is delocalized over many hydrogen molecules in a manner similar to a Frenkel exciton. It has a long coherence lifetime, so we have chosen solid p-H2 as our first target in the condensed phase. We shine a time-delayed pair of femtosecond laser pulses on p-H2 to generate vibrons. Their interference results in modulation of the amplitude of their superposition. Scanning the interpulse delay on the attosecond time scale gives a high interferometric contrast, which demonstrates the possibility of using solid p-H2 as a carrier of information encoded in the vibrons. In the second example, we have controlled the terahertz collective phonon motion, called a "coherent phonon", of a single crystal of bismuth. We employ an intensity-modulated laser pulse, whose temporal envelope is modulated with terahertz frequency by overlap of two positively chirped laser pulses with their adjustable time delay. This modulated laser pulse is shined on the bismuth crystal to excite its two orthogonal phonon modes. Their relative amplitudes are controlled by tuning the delay between the two chirped pulses on the attosecond time scale. Two-dimensional atomic motion in the crystal is thus controlled arbitrarily. The method is based on the simple, robust, and universal concept that in any physical system, two-dimensional particle motion is decomposed into two orthogonal one-dimensional motions, and thus, it is applicable to a variety of condensed matter systems. In the third example, the double-pulse interferometry used for solid p-H2 has been applied to many-body electronic wave functions of an ensemble of ultracold rubidium Rydberg atoms, hereafter called a "strongly correlated ultracold Rydberg gas". This has allowed the observation and control of many-body electron dynamics of more than 40 Rydberg atoms interacting with each other. This new combination of ultrafast coherent control and ultracold atoms offers a versatile platform to precisely observe and manipulate nonequilibrium dynamics of quantum many-body systems on the ultrashort time scale. These three examples are digested in this Account.

Characterization of Functional Primary Cilia in Human Induced Pluripotent Stem Cell-Derived Neurons.

Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Neurons differentiated from hiPSCs may be promising tools to develop novel treatment methods for various neurological diseases. However, the detailed process underlying functional maturation of hiPSC-derived neurons remains poorly understood. Here, we analyze the developmental architecture of hiPSC-derived cortical neurons, iCell GlutaNeurons, focusing on the primary cilium, a single sensory organelle that protrudes from the surface of most growth-arrested vertebrate cells. To characterize the neuronal cilia, cells were cultured for various periods and evaluated immunohistochemically by co-staining with antibodies against ciliary markers Arl13b and MAP2. Primary cilia were detected in neurons within days, and their prevalence and length increased with increasing days in culture. Treatment with the mood stabilizer lithium led to primary cilia length elongation, while treatment with the orexigenic neuropeptide melanin-concentrating hormone caused cilia length shortening in iCell GlutaNeurons. The present findings suggest that iCell GlutaNeurons develop neuronal primary cilia together with the signaling machinery for regulation of cilia length. Our approach to the primary cilium as a cellular antenna can be useful for both assessment of neuronal maturation and validation of pharmaceutical agents in hiPSC-derived neurons.

Attosecond Control of Restoration of Electronic Structure Symmetry.

Laser pulses can break the electronic structure symmetry of atoms and molecules by preparing a superposition of states with different irreducible representations. Here, we discover the reverse process, symmetry restoration, by means of two circularly polarized laser pulses. The laser pulse for symmetry restoration is designed as a copy of the pulse for symmetry breaking. Symmetry restoration is achieved if the time delay is chosen such that the superposed states have the same phases at the temporal center. This condition must be satisfied with a precision of a few attoseconds. Numerical simulations are presented for the C_{6}H_{6} molecule and ^{87}Rb atom. The experimental feasibility of symmetry restoration is demonstrated by means of high-contrast time-dependent Ramsey interferometry of the ^{87}Rb atom.

BDNF Reduces eEF2 Phosphorylation and Enhances Novel Protein Synthesis in the Growth Cones of Dorsal Root Ganglia Neurons.

The local translation, which is regulated by extracellular stimuli such as guidance molecules, in growth cones of neurons provides a molecular mechanism for axonal development. In this study, we performed immunocytochemistry together with atomic force microscopy to investigate the localization of ribosomal proteins in the growth cones of rat dorsal root ganglion (DRG) neurons. The immunoreactivity of ribosomal protein P0/1/2 and S6, and novel protein synthesis were observed in the central, sterically bulky region of growth cones. Brain derived neurotrophic factor (BDNF) reduced the eEF2 phosphorylation, indicating its activation, and enhanced protein synthesis within 30 min. The effects of BDNF were completely inhibited by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR). These results indicated that BDNF rapidly activates translation and enhances novel protein synthesis in growth cones of DRG though the mTOR signaling.

Epidermal growth factor signals attenuate phenotypic and functional development of neocortical GABA neurons.

Phenotypic development of neocortical GABA neurons is highly plastic and promoted by various neurotrophic factors such as neuregulin-1. A subpopulation of GABA neurons expresses not only neuregulin receptor (ErbB4) but also epidermal growth factor (EGF) receptor (ErbB1) during development, but the neurobiological action of EGF on this cell population is less understood than that of neuregulin-1. Here, we examined the effects of exogenous EGF on immature GABA neurons both in culture and in vivo and also explored physiological consequences in adults. We prepared low density cultures from the neocortex of rat embryos and treated neocortical neurons with EGF. EGF decreased protein levels of glutamic acid decarboxylases (GAD65 and GAD67), and EGF influences on neuronal survival and glial proliferation were negligible or limited. The EGF treatment also diminished the frequency of miniature inhibitory postsynaptic currents (mIPSCs). In vivo administration of EGF to mouse pups reproduced the above GABAergic phenomena in neocortical culture. In EGF-injected postnatal mice, GAD- and parvalbumin-immunoreactivities were reduced in the frontal cortex. In addition, postnatal EGF treatment decreased mIPSC frequency in, and the density of, GABAergic terminals on pyramidal cells. Although these phenotypic influences on GABA neurons became less marked during development, it later resulted in the reduced ß- and γ-powers of sound-evoked electroencephalogram in adults, which is regulated by parvalbumin-positive GABA neurons and implicated in the schizophrenia pathophysiology. These findings suggest that, in contrast to the ErbB4 ligand of neuregulin-1, the ErbB1 ligand of EGF exerts unique maturation-attenuating influences on developing cortical GABAergic neurons.

Advanced glycation end products induce brain-derived neurotrophic factor release from human platelets through the Src-family kinase activation.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) exerts beneficial effects not only on diabetic neuropathies but also on cardiovascular injury. There is argument regarding the levels of serum BDNF in patients with diabetes mellitus (DM). Because BDNF in peripheral blood is rich in platelets, this may represent dysregulation of BDNF release from platelets. Here we focused on advanced glycation end products (AGEs), which are elevated in patients with DM and have adverse effects on cardiovascular functions. The aim of this study is to elucidate the role of AGEs in the regulation of BDNF release from human platelets. METHODS: Platelets collected from peripheral blood of healthy volunteers were incubated with various concentrations of AGE (glycated-BSA) at 37 °C for 5 min with or without BAPTA-AM, a cell permeable Ca2+ chelator, or PP2, a potent inhibitor of Src family kinases (SFKs). Released and cellular BDNF were measured by ELISA and calculated. Phosphorylation of Src and Syk, a downstream kinase of SFKs, in stimulated platelets was examined by Western blotting and immunoprecipitation. RESULTS: AGE induced BDNF release from human platelets in a dose-dependent manner, which was dependent on intracellular Ca2+ and SFKs. We found that AGE induced phosphorylation of Src and Syk. CONCLUSIONS: AGE induces BDNF release from human platelets through the activation of the Src-Syk-(possibly phospholipase C)-Ca2+ pathway. Considering the toxic action of AGEs and the protective roles of BDNF, it can be hypothesized that AGE-induced BDNF release is a biological defense system in the early phase of diabetes. Chronic elevation of AGEs may induce depletion or downregulation of BDNF in platelets during the progression of DM.

Somatic Mutations in the MTOR gene cause focal cortical dysplasia type IIb.

OBJECTIVE: Focal cortical dysplasia (FCD) type IIb is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, and balloon cells. It has been suggested that FCDs are caused by somatic mutations in cells in the developing brain. Here, we explore the possible involvement of somatic mutations in FCD type IIb. METHODS: We collected a total of 24 blood-brain paired samples with FCD, including 13 individuals with FCD type IIb, 5 with type IIa, and 6 with type I. We performed whole-exome sequencing using paired samples from 9 of the FCD type IIb subjects. Somatic MTOR mutations were identified and further investigated using all 24 paired samples by deep sequencing of the entire gene's coding region. Somatic MTOR mutations were confirmed by droplet digital polymerase chain reaction. The effect of MTOR mutations on mammalian target of rapamycin (mTOR) kinase signaling was evaluated by immunohistochemistry and Western blotting analyses of brain samples and by in vitro transfection experiments. RESULTS: We identified four lesion-specific somatic MTOR mutations in 6 of 13 (46%) individuals with FCD type IIb showing mutant allele rates of 1.11% to 9.31%. Functional analyses showed that phosphorylation of ribosomal protein S6 in FCD type IIb brain tissues with MTOR mutations was clearly elevated, compared to control samples. Transfection of any of the four MTOR mutants into HEK293T cells led to elevated phosphorylation of 4EBP, the direct target of mTOR kinase. INTERPRETATION: We found low-prevalence somatic mutations in MTOR in FCD type IIb, indicating that activating somatic mutations in MTOR cause FCD type IIb.

Cell surface expression of the major amyloid-ß peptide (Aß)-degrading enzyme, neprilysin, depends on phosphorylation by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) and dephosphorylation by protein phosphatase 1a.

Neprilysin is one of the major amyloid-ß peptide (Aß)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aß imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aß levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aß levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aß levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aß in the brain.

AMP-activated protein kinase counteracts brain-derived neurotrophic factor-induced mammalian target of rapamycin complex 1 signaling in neurons.

Growth factors and nutrients, such as amino acids and glucose, regulate mammalian target of rapamycin complex 1 (mTORC1) signaling and subsequent translational control in a coordinated manner. Brain-derived neurotrophic factor (BDNF), the most prominent neurotrophic factor in the brain, activates mTORC1 and induces phosphorylation of its target, p70S6 kinase (p70S6K), at Thr389 in neurons. BDNF also increases mammalian target of rapamycin-dependent novel protein synthesis in neurons. Here, we report that BDNF-induced p70S6K activation is dependent on glucose, but not amino acids, sufficiency in cultured cortical neurons. AMP-activated protein kinase (AMPK) is the molecular background to this specific nutrient dependency. Activation of AMPK, which is induced by glucose deprivation, treatment with pharmacological agents such as 2-deoxy-D-glucose, metformin, and 5-aminoimidazole-4-carboxamide ribonucleoside or forced expression of a constitutively active AMPKα subunit, counteracts BDNF-induced phosphorylation of p70S6K and enhanced protein synthesis in cortical neurons. These results indicate that AMPK inhibits the effects of BDNF on mTORC1-mediated translation in neurons.

Axonal mRNA binding of hnRNP A/B is crucial for axon targeting and maturation of olfactory sensory neurons.

Spatiotemporal control of gene expression is important for neural development and function. Here, we show that heterogeneous nuclear ribonucleoprotein (hnRNP) A/B is highly expressed in developing olfactory sensory neurons (OSNs), and its knockout results in reduction in mature OSNs and aberrant targeting of OSN axons to the olfactory bulb. RNA immunoprecipitation analysis reveals that hnRNP A/B binds to a group of mRNAs that are highly related to axon projections and synapse assembly. Approximately 11% of the identified hnRNP A/B targets, including Pcdha and Ncam2, encode cell adhesion molecules. In Hnrnpab knockout mice, PCDHA and NCAM2 levels are significantly reduced at the axon terminals of OSNs. Furthermore, deletion of the hnRNP A/B-recognition motif in the 3' UTR of Pcdha leads to impaired PCDHA expression at the OSN axon terminals. Therefore, we propose that hnRNP A/B facilitates OSN maturation and axon projection by regulating the local expression of its target genes at axon terminals.

Periventricular nodular heterotopia functionally couples with the overlying hippocampus.

Patients with periventricular nodular heterotopia (PVNH) often have severe epilepsy. However, it is unclear how the heterotopia contributes to epileptogenesis. Recently, electrophysiologic studies using intraoperative depth electrodes have indicated that interaction between the heterotopia and overlying cortex is crucial for seizure onset. We performed an in vitro physiologic study using slices of resected brain from a 22-year-old man with PVNH, who manifested medically refractory mesial temporal lobe epilepsy. Preoperative evaluation indicated that the right mesial temporal structure and PVNH were the epileptogenic focus. The resected tissue was immediately immersed in cold artificial cerebrospinal fluid, and then slices of the brain tissue including the heterotopic nodules and overlying hippocampus were prepared. We electrically stimulated the incubated slices, and the elicited neural activities were analyzed as changes in the flavoprotein fluorescence signals. When we stimulated either the heterotopic nodule or the overlying hippocampus, clear functional coupling of neural activities between these structures was observed. The coupling response evoked by stimulation of the subiculum and developing within the heterotopic nodule was enhanced by application of bicuculline. Therefore, activities of the hippocampus and the nodule are closely correlated.

Novel Repositioning Therapy for Drug-Resistant Glioblastoma: In Vivo Validation Study of Clindamycin Treatment Targeting the mTOR Pathway and Combination Therapy with Temozolomide.

Multimodal therapy including surgery, radiation treatment, and temozolomide (TMZ) is performed on glioblastoma (GBM). However, the prognosis is still poor and there is an urgent need to develop effective treatments to improve survival. Molecular biological analysis was conducted to examine the signal activation patterns in GBM specimens and remains an open problem. Advanced macrolides, such as azithromycin, reduce the phosphorylation of p70 ribosomal protein S6 kinase (p70S6K), a downstream mammalian target of rapamycin (mTOR) effector, and suppress the proliferation of T-cells. We focused on its unique profile and screened for the antitumor activity of approved macrolide antibiotics. Clindamycin (CLD) reduced the viability of GBM cells in vitro. We assessed the effects of the candidate macrolide on the mTOR pathway through Western blotting. CLD attenuated p70S6K phosphorylation in a dose-dependent manner. These effects on GBM cells were enhanced by co-treatment with TMZ. Furthermore, CLD inhibited the expression of the O6-methylguanine-DNA methyltransferase (MGMT) protein in cultured cells. In the mouse xenograft model, CLD and TMZ co-administration significantly suppressed the tumor growth and markedly decreased the number of Ki-67 (clone MIB-1)-positive cells within the tumor. These results suggest that CLD suppressed GBM cell growth by inhibiting mTOR signaling. Moreover, CLD and TMZ showed promising synergistic antitumor activity.

Qualitative and quantitative re-evaluation of epidermal growth factor-ErbB1 action on developing midbrain dopaminergic neurons in vivo and in vitro: target-derived neurotrophic signaling (Part 1).

Although epidermal growth factor (EGF) receptor (ErbB1) is implicated in Parkinson's disease and schizophrenia, the neurotrophic action of ErbB1 ligands on nigral dopaminergic neurons remains controversial. Here, we ascertained colocalization of ErbB1 and tyrosine hydroxylase (TH) immunoreactivity and then characterized the neurotrophic effects of ErbB1 ligands on this cell population. In mesencephalic culture, EGF and glial-derived neurotrophic factor (GDNF) similarly promoted survival and neurite elongation of dopaminergic neurons and dopamine uptake. The EGF-promoted dopamine uptake was not inhibited by GDNF-neutralizing antibody or TrkB-Fc, whereas EGF-neutralizing antibody fully blocked the neurotrophic activity of the conditioned medium that was prepared from EGF-stimulated mesencephalic cultures. The neurotrophic action of EGF was abolished by ErbB1 inhibitors and genetic disruption of erbB1 in culture. In vivo administration of ErbB1 inhibitors to rat neonates diminished TH and dopamine transporter (DAT) levels in the striatum and globus pallidus but not in the frontal cortex. In parallel, there was a reduction in the density of dopaminergic varicosities exhibiting intense TH immunoreactivity. In agreement, postnatal erbB1-deficient mice exhibited similar decreases in TH levels. Although neurotrophic supports to dopaminergic neurons are redundant, these results confirm that ErbB1 ligands contribute to the phenotypic and functional development of nigral dopaminergic neurons.

Dopamine-dependent ectodomain shedding and release of epidermal growth factor in developing striatum: target-derived neurotrophic signaling (Part 2).

Epidermal growth factor (EGF) and structurally related peptides promote neuronal survival and the development of midbrain dopaminergic neurons; however, the regulation of their production has not been fully elucidated. In this study, we found that the treatment of striatal cells with dopamine agonists enhances EGF release both in vivo and in vitro. We prepared neuron-enriched and non-neuronal cell-enriched cultures from the striatum of rat embryos and challenged those with various neurotransmitters or dopamine receptor agonists. Dopamine and a dopamine D(1) -like receptor agonist (SKF38393) triggered EGF release from neuron-enriched cultures in a dose-dependent manner. A D(2) -like agonist (quinpirole) increased EGF release only from non-neuronal cell-enriched cultures. The EGF release from striatal neurons and non-neuronal cells was concomitant with ErbB1 phosphorylation and/or with the activation of a disintegrin and metalloproteinase and matrix metalloproteinase. The EGF release from neurons was attenuated by an a disintegrin and metalloproteinase/matrix metalloproteinase inhibitor, GM6001, and a calcium ion chelator, BAPTA/AM. Transfection of cultured striatal neurons with alkaline phosphatase-tagged EGF precursor cDNA confirmed that dopamine D(1) -like receptor stimulation promoted both ectodomain shedding of the precursor and EGF release. Therefore, the activation of striatal dopamine receptors induces shedding and release of EGF to provide a retrograde neurotrophic signal to midbrain dopaminergic neurons.