Pallidal Hyperdopaminergic Innervation Underlying D2 Receptor-Dependent Behavioral Deficits in the Schizophrenia Animal Model Established by EGF

Epidermal growth factor (EGF) is one of the ErbB receptor ligands implicated in schizophrenia neuropathology as well as in dopaminergic development. Based on the immune inflammatory hypothesis for schizophrenia, neonatal rats are exposed to this cytokine and later develop neurobehavioral abnormality such as prepulse inhibition (PPI) deficit. Here we found that the EGF-treated rats exhibited persistent increases in tyrosine hydroxylase levels and dopamine content in the globus pallidus. Furthermore, pallidal dopamine release was elevated in EGF-treated rats, but normalized by subchronic treatment with risperidone concomitant with amelioration of their PPI deficits. To evaluate pathophysiologic roles of the dopamine abnormality, we administered reserpine bilaterally to the globus pallidus to reduce the local dopamine pool. Reserpine infusion ameliorated PPI deficits of EGF-treated rats without apparent aversive effects on locomotor activity in these rats. We also administered dopamine D1-like and D2-like receptor antagonists (SCH23390 and raclopride) and a D2-like receptor agonist (quinpirole) to the globus pallidus and measured PPI and bar-hang latencies. Raclopride (0.5 and 2.0 µg/site) significantly elevated PPI levels of EGF-treated rats, but SCH23390 (0.5 and 2.0 µg/site) had no effect. The higher dose of raclopride induced catalepsy-like changes in control animals but not in EGF-treated rats. Conversely, local quinpirole administration to EGF-untreated control rats induced PPI deficits and anti-cataleptic behaviors, confirming the pathophysiologic role of the pallidal hyperdopaminergic state. These findings suggest that the pallidal dopaminergic innervation is vulnerable to circulating EGF at perinatal and/or neonatal stages and has strong impact on the D2-like receptor-dependent behavioral deficits relevant to schizophrenia

ErbB2 Dephosphorylation and Anti-Proliferative Effects of Neuregulin-1 in ErbB2-Overexpressing Cells; Re-evaluation of Their Low-Affinity Interaction

Neuregulin-1 binds to ErbB3 and ErbB4 and regulates cancer proliferation and differentiation. Neuregulin-1 had been suggested to also react with ErbB2, but this argument becomes controversial. Here, we re-evaluated the cellular responses and ErbB2 interaction of neuregulin-1 in ErbB2 overexpressing cell lines. In a competitive ligand-binding assay, we detected significant replacement of [35S]-labeled neuregulin-1 with nano molar ranges of cold neuregulin-1 in L929 cells expressing ErbB2 alone and SKOV3 cells carrying sulf-1 cDNA but not in these parental cells. The concentration of neuregulin-1 significantly decreased thymidine incorporation and phosphorylation of ErbB2 (Tyr877, Tyr1396, and Tyr1121) in ErbB2-overexpressing cancer cells as well as in L929 cells expressing ErbB2. A crosslinking assay ascertained the presence of neuregulin-1 immunoreactivity in the ErbB2 immune complexes of L929 expressing ErbB2 alone. These results suggest that the higher concentrations of neuregulin-1 exert an anti-oncogenic activity to attenuate ErbB2 auto-phosphorylation potentially through its low-affinity interaction with ErbB2

Neuropathologic Implication of Peripheral Neuregulin-1 and EGF Signals in Dopaminergic Dysfunction and Behavioral Deficits Relevant to Schizophrenia: Their Target Cells and Time Window

Neuregulin-1 and epidermal growth factor (EGF) are implicated in the pathogenesis of schizophrenia. To test the developmental hypothesis for schizophrenia, we administered these factors to rodent pups, juveniles, and adults and characterized neurobiological and behavioral consequences. These factors were also provided from their transgenes or infused into the adult brain. Here we summarize previous results from these experiments and discuss those from neuropathological aspects. In the neonatal stage but not the juvenile and adult stages, subcutaneously injected factors penetrated the blood-brain barrier and acted on brain neurons, which later resulted in persistent behavioral and dopaminergic impairments associated with schizophrenia. Neonatally EGF-treated animals exhibited persistent hyperdopaminergic abnormalities in the nigro-pallido-striatal system while neuregulin-1 treatment resulted in dopaminergic deficits in the corticolimbic dopamine system. Effects on GABAergic and glutamatergic systems were transient or limited. Even in the adult stage, intracerebral administration and transgenic expression of these factors produced similar but not identical behavioral impairments, although the effects of intracerebral administration were reversible. These findings suggest that dopaminergic development is highly vulnerable to circulating ErbB ligands in the pre- and perinatal stages. Once maldevelopment of the dopaminergic system is established during early development, dopamine-associating behavioral deficits become irreversible and manifest at postpubertal stages

Glutamate-dependent ectodomain shedding of neuregulin-1 type II precursors in rat forebrain neurons

<div><p>The neurotrophic factor neuregulin 1 (NRG1) regulates neuronal development, glial differentiation, and excitatory synapse maturation. NRG1 is synthesized as a membrane-anchored precursor and is then liberated by proteolytic processing or exocytosis. Mature NRG1 then binds to its receptors expressed by neighboring neurons or glial cells. However, the molecular mechanisms that govern this process in the nervous system are not defined in detail. Here we prepared neuron-enriched and glia-enriched cultures from embryonic rat neocortex to investigate the role of neurotransmitters that regulate the liberation/release of NRG1 from the membrane of neurons or glial cells. Using a two-site enzyme immunoassay to detect soluble NRG1, we show that, of various neurotransmitters, glutamate was the most potent inducer of NRG1 release in neuron-enriched cultures. NRG1 release in glia-enriched cultures was relatively limited. Furthermore, among glutamate receptor agonists, N-Methyl-D-Aspartate (NMDA) and kainate (KA), but not AMPA or tACPD, mimicked the effects of glutamate. Similar findings were acquired from analysis of the hippocampus of rats with KA-induced seizures. To evaluate the contribution of members of a disintegrin and metalloproteinase (ADAM) families to NRG1 release, we transfected primary cultures of neurons with cDNA vectors encoding NRG1 types I, II, or III precursors, each tagged with the alkaline phosphatase reporter. Analysis of alkaline phosphatase activity revealed that the NRG1 type II precursor was subjected to tumor necrosis factor-α-converting enzyme (TACE) / a Disintegrin And Metalloproteinase 17 (ADAM17) -dependent ectodomain shedding in a protein kinase C-dependent manner. These results suggest that glutamatergic neurotransmission positively regulates the ectodomain shedding of NRG1 type II precursors and liberates the active NRG1 domain in an activity-dependent manner.</p></div

Effects of neurotransmitters on NRG1 release from neuron-enriched rat cortical cultures.

<p>(A) Neuron-enriched cultures were treated with control vehicle (Cont), glutamate (Glu, 10 μM, 20 min), acetylcholine (Ach, 100 μM, 20 min), dopamine (DA, 30 μM, 20 min) or serotonin (5HT, 100 μM, 20 min), and PMA (1 μM, 30 min) on day 7 (DIV7). (B) Dose dependency of NRG1 release. Cultures were treated with 0, 10, 30, and 100 μM glutamate or acetylcholine (20 min). (C) Western blotting using anti-phospho-ErbB4 and anti-ErbB4 antibodies. Representative immunoblots are shown. The mean levels of phospho-ErbB4 and ErbB4 immunoreactivities in controls were defined as 100%. (D) Effects of glutamate receptor antagonists and acetylcholine receptor agonists on NRG1 release. Cultures were pretreated with AP5 and CNQX (50 μM and 10 μM, 20 min), then control vehicle (Cont), acetylcholine (Ach, 100 μM, 20 min), carbachole (Carba, 100 μM, 20 min), nicotine (Nico, 100 μM, 20 min), muscarine (Mus, 100 μM, 20 min), glutamate (Glu, 10 μM, 20 min), or PMA (1 μM, 30 min). NRG1 concentrations in culture supernatants were measured using an ELISA. Data represent the mean ± SD (four sister cultures, each); *<i>p</i> < 0.05, **<i>p</i> < 0.01 vs. control vehicle.</p

Neuron–target interactions mediated by glutamate and the NRG1 type II isoform.

<p>Glutamate released from presynaptic terminals binds to postsynaptic glutamate receptors and activates ADAMs in neurons (e.g., ADAM17) via PKC. ADAMs then cleave the ectodomain of the NRG1 type II precursor and liberates mature NRG1 type II. Free NRG1 type II binds ErbB4 receptors in neurons or neighboring neurons, presumably leading to glutamatergic development, maturation, or both.</p

NRG1 release and ErbB4 phosphorylation following KA receptor activation <i>in vivo</i>.

<p>(A) Levels of soluble-free NRG1 in the hippocampus were determined in epileptic and control rats. (B) Total ADAM activity in hippocampal tissue lysates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174780#pone.0174780.g005" target="_blank">Fig 5</a>. (C) Crude hippocampal membranes were subjected to Western blotting using anti-phospho-ErbB4 and anti-ErbB4 antibodies. The mean levels of phospho-ErbB4 and ErbB4 immunoreactivities in controls were defined as 100%. Data represent mean ± SD (four rats per group); *<i>p</i> < 0.05, **<i>p</i> < 0.01, vs. saline-treated control group.</p

Glutamate receptor agonists increase type II NRG1 release.

<p>(A) After the transfection of cells with the NRG1 type II-AP expression vector, neuron-enriched cultures were treated with control vehicle (saline), glutamate (Glu, 10 μM), NMDA (100 μM), AMPA (100 μM), KA (100 μM), tAPCD (100 μM), or L-AP4 (50 μM) for 20 min as described above. The enzyme activities of the AP tag in conditioned medium and at the cell surface are displayed. (B, C) Effects of the glutamate receptor antagonists (AP5, CNQX) on AP activity in conditioned medium (B) and on the cell surface (C) are shown. Data represent the mean ± SD (four sister cultures each); *<i>p</i> < 0.05, **<i>p</i> < 0.01 vs. control vehicle.</p

Effects of botulinum toxin and an MMP inhibitor on NRG1 release.

<p>(A) Neuron-enriched cultures were pretreated with GM6001 (100 nM, 1 h), BoNT/A (100 nM, 3 h), or control vehicle prior to challenge with vehicle, glutamate (10 μM, 20 min), or PMA (1 μM, 30 min). NRG1 concentrations in culture supernatants were measured using ELISA. (B) Neuron-enriched cultures were pretreated with GM6001, AP5, or CNQX and then challenged with control vehicle, glutamate, NMDA, or PMA as described above. Cell lysates were subjected to Western blotting using anti-phospho-ErbB4 and anti-ErbB4 antibodies. Representative immunoblots are shown. Data represent the mean ± SD (four sister cultures each); **<i>p</i> < 0.01 vs. control vehicle.</p