Yuriko Iwakura

Influences of dopaminergic lesion on epidermal growth factor-ErbB signals in Parkinson's disease and its model: neurotrophic implication in nigrostriatal neurons.

Epidermal growth factor (EGF) is a member of a structurally related family containing heparin‐binding EGF‐like growth factor (HB‐EGF) and transforming growth factor alpha (TGFα) that exerts neurotrophic activity on midbrain dopaminergic neurons. To examine neurotrophic abnormality in Parkinsons disease (PD), we measured the protein content of EGF, TGFα, and HB‐EGF in post‐mortem brains of patients with Parkinsons disease and age‐matched control subjects. Protein levels of EGF and tyrosine hydroxylase were decreased in the prefrontal cortex and the striatum of patients. In contrast, HB‐EGF and TGFα levels were not significantly altered in either region. The expression of EGF receptors (ErbB1 and ErbB2, but not ErbB3 or ErbB4) was down‐regulated significantly in the same forebrain regions. The same phenomenon was mimicked in rats by dopaminergic lesions induced by nigral 6‐hydroxydopamine infusion. EGF and ErbB1 levels in the striatum of the PD model were markedly reduced on the lesioned side, compared with the control hemisphere. Subchronic supplement of EGF in the striatum of the PD model locally prevented the dopaminergic neurodegeration as measured by tyrosine hydroxylase immunoreactivity. These findings suggest that the neurotrophic activity of EGF is maintained by afferent signals of midbrain dopaminergic neurons and is impaired in patients with Parkinsons disease.

Brain-derived Neurotrophic Factor Regulates Surface Expression of α-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic Acid Receptors by Enhancing the N-Ethylmaleimide-sensitive Factor/GluR2 Interaction in Developing Neocortical Neurons

In hippocampal neurons, the exocytotic process of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors is known to depend on activation ofN-methyl-d-aspartate channels and its resultant Ca2+ influx from extracellular spaces. Here we found that brain-derived neurotrophic factor (BDNF) induced a rapid surface translocation of AMPA receptors in an activity-independent manner in developing neocortical neurons. The receptor translocation became evident within hours as monitored by [3H]AMPA binding and was resistant against ionotropic glutamate receptor antagonists as evidenced with surface biotinylation assay. This process required intracellular Ca2+ and was inhibited by the blockers of conventional exocytosis, brefeldin A, botulinum toxin B, andN-ethylmaleimide. To explore the translocation mechanism of individual AMPA receptor subunits, we utilized the human embryonic kidney (HEK) 293 cells carrying the BDNF receptor TrkB. After the single transfection of GluR2 cDNA or GluR1 cDNA into HEK/TrkB cells, BDNF triggered the translocation of GluR2 but not that of GluR1. Subsequent mutation analysis of GluR2 carboxyl-terminal region indicated that the translocation of GluR2 subunit in HEK293 cells involved its N-ethylmaleimide-sensitive factor-binding domain but not its PDZ-interacting site. Following co-transfection of GluR1 and GluR2 cDNAs, solid phase cell sorting revealed that GluR1 subunits were also able to translocate to the cell surface in response to BDNF. An immunoprecipitation assay confirmed that BDNF stimulation can enhance the interaction of GluR2 withN-ethylmaleimide-sensitive factor. These results reveal a novel role of BDNF in regulating the surface expression of AMPA receptors through a GluR2-NSF interaction.

Phenotypic down-regulation of glutamate receptor subunit GluR1 in Alzheimer's disease.

Glutamate receptors play crucial roles in cognition and memory. We have quantitated the protein levels of alpha-amino-isoxazolepropionic acid (AMPA)-type (GluR1) and N-methyl-D-aspartate-type (NMDAR1) glutamate receptors in postmortem brain tissues of patients with Alzheimers disease and age-matched controls using western blotting. The bolts carrying fully denatured proteins were probed with antibodies specific to their carboxyl terminus of these receptors. In Alzheimers disease, GluR1 levels were significantly decreased in the entorhinal cortex and dentate gyrus, but not in the motor cortex. In contrast, levels of NMDAR1 were not altered in the dentate gyrus, suggesting that GluR1 expression was specifically diminished in this structure that is known to be preserved histologically in patients. However, the results of immunocytochemical examination confirmed a previous controversial report: GluR1-immunoreactive structures were labeled rather intensely in the molecular layer of the dentate gyrus of Alzheimers patients. Interestingly, levels of a postsynaptic density protein named SAP97, which recognizes and potentially masks the epitope region of GluR1, was positively correlated with those of GluR1 protein in the control group, but not in the patient group. Thus, the enhanced GluR1-like staining in Alzheimers disease might be ascribed to the hampered interaction between SAP97 and GluR1 leading to epitope unmasking of GluR1 on tissue sections. These findings indicate that abnormal expressions of the AMPA receptor and its interacting PSD molecule are associated with Alzheimers disease and implicated in pathophysiology of this disease.

Dopamine D1 Receptor-induced Signaling through TrkB Receptors in Striatal Neurons

In addition to its role as a neurotransmitter, dopamine can stimulate neurite outgrowth and morphological effects upon primary neurons. To investigate the signal transduction mechanisms used by dopamine in developing striatal neurons, we focused upon the effects of activating the dopamine D1 receptor. Using the D1 receptor agonist SKF38393, we found that Trk neurotrophin receptors were activated in embryonic day 18 striatal neurons. K-252a, a Trk tyrosine kinase inhibitor, and a dopamine D1 receptor antagonist could block the effects of SKF38393. The increase in TrkB phosphorylation was not the result of increased neurotrophin production. Induction of TrkB activity by SKF38393 was accompanied by the phosphorylation of several Trk signaling proteins, including phospholipase Cγ, Akt, and MAPK. Biotinylation experiments followed by immunostaining by phospho-TrkB-specific antibodies indicated that the mechanism involved increased TrkB surface expression by dopamine D1 receptor activation. This increase in cell surface TrkB expression was dependent upon an increase in intracellular Ca2+. These results indicate that stimulation of dopamine D1 receptors can be coupled to the neurotrophin receptor signaling to mediate the effects of dopamine upon striatal neurons.

ErbB1-4-dependent EGF/neuregulin signals and their cross talk in the central nervous system: pathological implications in schizophrenia and Parkinson's disease

Ligands for ErbB1-4 receptor tyrosine kinases, such as epidermal growth factor (EGF) and neuregulins, regulate brain development and function. Thus, abnormalities in their signaling are implicated in the etiology or pathology of schizophrenia and Parkinsons disease. Among the ErbB receptors, ErbB1, and ErbB4 are expressed in dopamine and GABA neurons, while ErbB1, 2, and/or 3 are mainly present in oligodendrocytes, astrocytes, and their precursors. Thus, deficits in ErbB signaling might contribute to the neurological and psychiatric diseases stemming from these cell types. By incorporating the latest cancer molecular biology as well as our recent progress, we discuss signal cross talk between the ErbB1-4 subunits and their neurobiological functions in each cell type. The potential contribution of virus-derived cytokines (virokines) that mimic EGF and neuregulin-1 in brain diseases are also discussed.

Neuregulin-1 signals from the periphery regulate AMPA receptor sensitivity and expression in GABAergic interneurons in developing neocortex.

Neuregulin-1 (NRG1) signaling is thought to contribute to both neuronal development and schizophrenia neuropathology. Here, we describe the developmental effects of excessive peripheral NRG1 signals on synaptic activity and AMPA receptor expression of GABAergic interneurons in postnatal rodent neocortex. A core peptide common to all NRG1 variants (eNRG1) was subcutaneously administered to mouse pups. Injected eNRG1 penetrated the blood–brain barrier and activated ErbB4 NRG1 receptors in the neocortex, in which ErbB4 mRNA is predominantly expressed by parvalbumin-positive GABAergic interneurons. We prepared neocortical slices from juvenile mice that were receiving eNRG1 subchronically and recorded inhibitory synaptic activity from layer V pyramidal neurons. Postnatal eNRG1 treatment significantly enhanced polysynaptic IPSCs, although monosynaptic IPSCs were not affected. Examination of excitatory inputs to parvalbumin-containing GABAergic interneurons revealed that eNRG1 treatment significantly increased AMPA-triggered inward currents and the amplitudes and frequencies of miniature EPSCs (mEPSCs). Similar effects on mEPSCs were observed in mice treated with a soluble, full-length form of NRG1 type I. Consistent with the electrophysiologic data, expression of the AMPA receptor GluA1 (i.e., GluR1, GluRA) was upregulated in the postsynaptic density/cytoskeletal fraction prepared from eNRG1-treated mouse neocortices. Cortical GABAergic neurons cultured with eNRG1 exhibited a significant increase in surface GluA1 immunoreactivity at putative synaptic sites on their dendrites. These results indicate that NRG1 circulating in the periphery influences postnatal development of synaptic AMPA receptor expression in cortical GABAergic interneurons and may play a role in conditions characterized by GABA-associated neuropathologic processes.

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)

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07287.x

Transforming growth factor alpha attenuates the functional expression of AMPA receptors in cortical GABAergic neurons.

In the developing neocortex, brain-derived neurotrophic factor (BDNF) exerts a trophic activity to increase the expression and channel activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits. Here, we demonstrate that the epidermal growth factor (EGF) receptor (ErbB1) ligands exert the opposite biological activity in cultured neocortical neurons. Subchronic stimulation of ErbB1 with transforming growth factor alpha (TGFalpha), EGF, or heparin-binding EGF (HB-EGF) down-regulated protein expression of the GluR1 AMPA receptor subunit in cultured neocortical neurons. In agreement, TGFalpha treatment decreased the Bmax of [3H] AMPA binding and GluR1 mRNA levels. Immunocytochemistry revealed that the decrease in GluR1 was most pronounced in multipolar GABAergic neurons. To examine the physiological consequences, we recorded AMPA-evoked currents as well as miniature excitatory postsynaptic currents in morphologically identified putative GABAergic neurons in culture. Subchronic TGFalpha treatment decreased AMPA-triggered currents as well as the amplitude and frequency of miniature excitatory postsynaptic currents. An ErbB1 tyrosine kinase inhibitor, PD153035, inhibited the TGFalpha effect. Moreover, TGFalpha counteracted the neurotrophic activity of BDNF on AMPA receptor expression. Co-application of TGFalpha with BDNF blocked the BDNF-triggered up-regulation of AMPA receptor expression and currents. These observations reveal a negative regulatory activity of the ErbB1 ligand, TGFalpha, which reduces the input sensitivity of cortical GABAergic neurons to attenuate their inhibitory function.

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.

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

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07295.x