Sustained ErbB Activation Causes Demyelination and Hypomyelination by Driving Necroptosis of Mature Oligodendrocytes and Apoptosis of Oligodendrocyte Precursor Cells.

Oligodendrocytes are vulnerable to genetic and environmental insults and its injury leads to demyelinating diseases. The roles of ErbB receptors in maintaining the CNS myelin integrity are largely unknown. Here, we overactivate ErbB receptors that mediate signaling of either neuregulin (NRG) or epidermal growth factor (EGF) family growth factors and found their synergistic activation caused deleterious outcomes in white matter. Sustained ErbB activation induced by the tetracycline-dependent mouse tool Plp-tTA resulted in demyelination, axonal degeneration, oligodendrocyte precursor cell (OPC) proliferation, astrogliosis, and microgliosis in white matter. Moreover, there was hypermyelination before these inflammatory pathologic events. In contrast, sustained ErbB activation induced by another tetracycline-dependent mouse tool Sox10+/rtTA caused hypomyelination in the corpus callosum and optic nerve, which appeared to be a developmental deficit and did not associate with OPC regeneration, astrogliosis, or microgliosis. By tracing the differentiation states of cells expressing tetracycline-controlled transcriptional activator (tTA)/reverse tTA (rtTA)-dependent transgene or pulse-labeled reporter proteins in vitro and in vivo, we found that Plp-tTA targeted mainly mature oligodendrocytes (MOs), whereas Sox10+/rtTA targeted OPCs and newly-formed oligodendrocytes (NFOs). The distinct phenotypes of mice with ErbB overactivation induced by Plp-tTA and Sox10+/rtTA consolidated their nonoverlapping targeting preferences in the oligodendrocyte lineage, and enabled us to demonstrate that ErbB overactivation in MOs induced necroptosis that caused inflammatory demyelination, whereas in OPCs induced apoptosis that caused noninflammatory hypomyelination. Early interference with aberrant ErbB activation ceased oligodendrocyte deaths and restored myelin development in both mice. This study suggests that aberrant ErbB activation is an upstream pathogenetic mechanism of demyelinating diseases, providing a potential therapeutic target.SIGNIFICANCE STATEMENT Primary oligodendropathy is one of the etiologic mechanisms for multiple sclerosis, and oligodendrocyte necroptosis is a pathologic hallmark in the disease. Moreover, the demyelinating disease is now a broad concept that embraces schizophrenia, in which white matter lesions are an emerging feature. ErbB overactivation has been implicated in schizophrenia by genetic analysis and postmortem studies. This study suggests the etiologic implications of ErbB overactivation in myelin pathogenesis and elucidates the pathogenetic mechanisms.

Stage-dependent regulation of oligodendrocyte development and enhancement of myelin repair by dominant negative Master-mind 1 protein.

Notch signaling has been implicated in the inhibition of oligodendrocyte differentiation and myelin gene expression during early development. However, inactivation of a particular Notch or Hes gene only produces a mild phenotype in oligodendrocyte development possibly due to the functional redundancies among closely related family members. To uncover the full role of Notch signaling in myelin development and regeneration, we generated the Sox10rtTA/+ ; TetO-dnMAML1 double transgenic mice in which expression of dominant negative Master-mind 1 (dnMAML1) gene can be selectively induced in oligodendrocyte precursor cells (OPCs) for complete blockade of Notch signaling. It is found that dnMAML1 expression leads to robust precocious OL differentiation and premature axonal myelination in the spinal cord, possibly by upregulating Nkx2.2 and downregulating Pdgfra expression. Unexpectedly, at late embryonic stages, dnMAML1 expression dramatically increased the number of OPCs, indicating a stage-dependent effect of Notch signaling on OPC proliferation. In addition, dnMAML1 also significantly enhances axonal remyelination following chemical-induced demyelination, providing a promising therapeutic target for lesion repair in demyelinating disease.

Role of Erbin in ErbB2-dependent breast tumor growth.

ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2), a receptor tyrosine kinase of the ErbB family, is overexpressed in around 25% of breast cancers. In addition to forming a heterodimer with other ErbB receptors in response to ligand stimulation, ErbB2 can be activated in a ligand-independent manner. We report here that Erbin, an ErbB2-interacting protein that was thought to act as an antitumor factor, is specifically expressed in mammary luminal epithelial cells and facilitates ErbB2-dependent proliferation of breast cancer cells and tumorigenesis in MMTV-neu transgenic mice. Disruption of their interaction decreases ErbB2-dependent proliferation, and deletion of the PDZ domain in Erbin hinders ErbB2-dependent tumor development in MMTV-neu mice. Mechanistically, Erbin forms a complex with ErbB2, promotes its interaction with the chaperon protein HSP90, and thus prevents its degradation. Finally, ErbB2 and Erbin expression correlates in human breast tumor tissues. Together, these observations establish Erbin as an ErbB2 regulator for breast tumor formation and progression.

Erbin is required for myelination in regenerated axons after injury.

Neuregulin 1 (NRG1) is an axon-derived factor that is critical for Schwann cell (SC) development and myelinogenesis in a manner dependent on transmembrane tyrosine kinases ErbB2 and ErbB3. Recent studies suggest that NRG1 signaling plays a role in remyelination of regenerated nerves after injury. In this study, we investigated the role of Erbin, a protein that interacts with ErbB2 in remyelination of injured nerves. We show that Erbin expression increased dramatically in injured nerves. Myelinated axons were fewer, and g-ratios of those that were myelinated were increased in erbin(-/-) mice, which were impaired in functional recovery from nerve injury. These results indicate a necessary role of Erbin in remyelination of regenerating axons. Erbin ablation had little effect on numbers of BrdU-labeled and TUNEL-labeled SCs, suggesting mechanisms independent of altered proliferation or apoptosis. We demonstrated that Erbin mutant mice were impaired in raising or maintaining the levels of ErbB2 and in producing NRG1 in axons. Together, these observations demonstrate that Erbin is required for remyelination of regenerated axons after injury, probably by regulating ErbB2 and NRG1 levels, identifying a novel player in regulating remyelination.

Neuregulin 1 promotes excitatory synapse development and function in GABAergic interneurons.

Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD-95 puncta and the frequency and amplitude of miniature EPSCs (mEPSCs) in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced frequency and amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in excitatory synaptogenesis in interneurons. Together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism.

Erbin regulates NRG1 signaling and myelination.

Neuregulin 1 (NRG1) plays a critical role in myelination. However, little is known about regulatory mechanisms of NRG1 signaling. We show here that Erbin, a protein that contains leucine-rich repeats (LRR) and a PSD95-Dlg-Zol (PDZ) domain and that interacts specifically with ErbB2, is necessary for NRG1 signaling and myelination of peripheral nervous system (PNS). In Erbin null mice, myelinated axons were hypomyelinated with reduced expression of P0, a marker of mature myelinating Schwann cells (SCs), whereas unmyelinated axons were aberrantly ensheathed in Remak bundles, with increased numbers of axons in the bundles and in pockets. The morphological deficits were associated with decreased nerve conduction velocity and increased sensory threshold to mechanistic stimulation. These phenotypes were duplicated in erbin(DeltaC/DeltaC) mice, in which Erbin lost the PDZ domain to interact with ErbB2. Moreover, ErbB2 was reduced at protein levels in both Erbin mutant sciatic nerves, and ErbB2 became unstable and NRG1 signaling compromised when Erbin expression was suppressed. These observations indicate a critical role of Erbin in myelination and identify a regulatory mechanism of NRG1 signaling. Our results suggest that Erbin, via the PDZ domain, binds to and stabilizes ErbB2, which is necessary for NRG1 signaling that has been implicated in tumorigenesis, heart development, and neural function.

Neuregulin-1 enhances depolarization-induced GABA release.

Neuregulin-1 (NRG1), a regulator of neural development, has been shown to regulate neurotransmission at excitatory synapses. Although ErbB4, a key NRG1 receptor, is expressed in glutamic acid decarboxylase (GAD)-positive neurons, little is known about its role in GABAergic transmission. We show that ErbB4 is localized at GABAergic terminals of the prefrontal cortex. Our data indicate a role of NRG1, both endogenous and exogenous, in regulation of GABAergic transmission. This effect was blocked by inhibition or mutation of ErbB4, suggesting the involvement of ErbB4. Together, these results indicate that NRG1 regulates GABAergic transmission via presynaptic ErbB4 receptors, identifying a novel function of NRG1. Because both NRG1 and ErbB4 have emerged as susceptibility genes of schizophrenia, these observations may suggest a mechanism for abnormal GABAergic neurotransmission in this disorder.

Interference of commissural connections through the genu of the corpus callosum specifically impairs sensorimotor gating.

White matter abnormalities in schizophrenic patients are characterized as regional tract-specific. Myelin loss at the genu of the corpus callosum (GCC) is one of the most consistent findings in schizophrenic patients across the different populations. We characterized the axons that pass through the GCC by stereotactically injecting an anterograde axonal tracing viral vector into the forceps minor of the corpus callosum in one hemisphere, and identified the homotopic brain structures that have commissural connections in the two hemispheres of the prefrontal cortex, including the anterior cingulate area, the prelimbic area, the secondary motor area, and the dorsal part of the agranular insular area, along with commissural connections with the primary motor area, caudoputamen, and claustrum. To investigate whether dysmyelination in these commissural connections is critical for the development of schizophrenia symptoms, we generated a mouse model with focal demyelination at the GCC by stereotactically injecting demyelinating agent lysolecithin into this site, and tested these mice in a battery of behavioral tasks that are used to model the schizophrenia-like symptom domains. We found that demyelination at the GCC influenced neither the social interest or mood state, nor the locomotive activity or motor coordination. Nevertheless, it specifically reduced the prepulse inhibition of acoustic startle that is a well-known measure of sensorimotor gating. This study advances our understanding of the pathophysiological contributions of the GCC-specific white matter lesion to the related disease, and demonstrates an indispensable role of interhemispheric communication between the frontal cortices for the top-down regulation of the sensorimotor gating.

Erbin in Amygdala Parvalbumin-Positive Neurons Modulates Anxiety-like Behaviors.

BACKGROUND: Anxiety disorders are the most common psychiatric diseases, affecting 28% of people worldwide within their lifetime. The excitation-inhibition imbalance in the amygdala is thought to be an underlying pathological mechanism; however, the cellular and molecular control of amygdala excitation-inhibition balance is largely unknown. METHODS: By using mice expressing chemogenetic activator or inhibitor channel in amygdala parvalbumin (PV) neurons, Erbin mutant mice, and mice with Erbin specifically knocked down in amygdala PV neurons, we systematically investigated the role of amygdala PV neurons and Erbin expressed therein in the pathogenesis of anxiety disorders using the combined approaches of immunohistochemistry, electrophysiology, and behavior. RESULTS: In naïve mice, chemogenetic inhibition of PV neurons produced anxiogenic effects, suggesting an essential role in the regulation of anxiety. In stressed mice with anxiety, excitatory postsynaptic responses on amygdala PV neurons were selectively diminished, accompanied by a decreased expression of Erbin specifically in amygdala PV neurons. Remarkably, both Erbin mutant mice and amygdala PV-specific Erbin knockdown mice exhibited impaired excitatory postsynaptic responses on amygdala PV neurons and increased anxiety-like behaviors. Furthermore, chemogenetic activation of amygdala PV neurons normalized anxiety behaviors in amygdala PV-specific Erbin knockdown mice and stressed mice. CONCLUSIONS: Together, these results demonstrate that Erbin in PV neurons is critical for maintaining the excitation-inhibition balance in the amygdala and reveal a novel pathophysiological mechanism for anxiety disorders.

Erbin controls dendritic morphogenesis by regulating localization of delta-catenin.

The LAP [leucine-rich and postsynaptic density-95/Discs large/zona occludens-1 (PDZ)] protein erbin and delta-catenin, a component of the cadherin-catenin cell adhesion complex, are highly expressed in neurons and associate through PDZ-mediated interaction, but have incompletely characterized neuronal functions. We show that short hairpin RNA-mediated knockdown of erbin and knockdown or genetic ablation of delta-catenin severely impaired dendritic morphogenesis in hippocampal neurons. Simultaneous loss of erbin and delta-catenin does not enhance severity of this phenotype. The dendritic phenotype observed after erbin depletion is rescued by overexpression of delta-catenin and requires a domain in delta-catenin that has been shown to regulate dendritic branching. Knockdown of delta-catenin cannot be rescued by overexpression of erbin, indicating that erbin is upstream of delta-catenin. delta-Catenin-null neurons have no alterations in global levels of active Rac1/RhoA. Knockdown of erbin results in alterations in localization of delta-catenin. These results suggest a critical role for erbin in regulating dendritic morphogenesis by maintaining appropriate localization of delta-catenin.

Low K+ promotes NF-kappaB/DNA binding in neuronal apoptosis induced by K+ loss.

Low intracellular K+ concentration ([K+]i) promotes apoptosis and blocking K+ loss prevents apoptosis, but the mechanism of action of low [K+]i remains unclear. Here, we show that low [K+]i increases NF-kappaB transcriptional activity by enhancing its binding to the promoter of target genes without affecting its activation and nuclear translocation in cortical neurons deprived of serum. Low K+ concentration promotes NF-kappaB/DNA binding through direct effects on the interaction of NF-kappaB dimers with DNA. Up-regulation of proapoptotic protein Bcl-XS and neuronal apoptosis induced by serum deprivation are blocked by inhibition and/or down-regulation of NF-kappaB and by prevention of K+ loss. Thus, a direct action of K+ on NF-kappaB/DNA binding regulates gene transcription related to neuronal apoptosis.

A role for ErbB signaling in the induction of reactive astrogliosis.

Reactive astrogliosis is a hallmark of many neurological disorders, yet its functions and molecular mechanisms remain elusive. Particularly, the upstream signaling that regulates pathological responses of astrocytes is largely undetermined. We used a mouse traumatic brain injury model to induce astrogliosis and revealed activation of ErbB receptors in reactive astrocytes. Moreover, cell-autonomous inhibition of ErbB receptor activity in reactive astrocytes by a genetic approach suppressed hypertrophic remodeling possibly through the regulation of actin dynamics. However, inhibiting ErbB signaling in reactive astrocytes did not affect astrocyte proliferation after brain injury, although it aggravated local inflammation. In contrast, active ErbB signaling in mature astrocytes of various brain regions in mice was sufficient to initiate reactive responses, reproducing characterized molecular and cellular features of astrogliosis observed in injured or diseased brains. Further, prevalent astrogliosis in the brain induced by astrocytic ErbB activation caused anorexia in animals. Therefore, our findings defined an unrecognized role of ErbB signaling in inducing reactive astrogliosis. Mechanistically, inhibiting ErbB signaling in reactive astrocytes prominently reduced Src and focal adhesion kinase (FAK) activity that is important for actin remodeling, although ErbB signaling activated multiple downstream signaling proteins. The discrepancies between the results from loss- and gain-of-function studies indicated that ErbB signaling regulated hypertrophy and proliferation of reactive astrocytes by different downstream signaling pathways. Our work demonstrated an essential mechanism in the pathological regulation of astrocytes and provided novel insights into potential therapeutic targets for astrogliosis-implicated diseases.

Isolation and culture of Schwann cells.

Primarily cultured Schwann cells are essential for the investigation of molecular mechanisms regulating proliferation, survival, differentiation, and myelination of Schwann cell and for the development of efficient transplantation for regeneration of injured spinal cord or peripheral nervous system. Here we describe a basic protocol for isolation and purification of primary Schwann cell from neonatal rat or mouse and discuss some modifications adapted to the culturing from adult nerves and optional methods for Schwann cell enrichment.

Erbin interacts with TARP γ-2 for surface expression of AMPA receptors in cortical interneurons.

Inhibitory neurons control the firing of glutamatergic neurons and synchronize brain activity. However, little is known about mechanisms of excitatory synapse formation in inhibitory neurons. Here we demonstrate that Erbin is specifically expressed in cortical inhibitory neurons. It localizes at excitatory synapses and regulates AMPA receptor (AMPAR) surface expression. Erbin mutation reduced mEPSCs and AMPAR currents specifically in parvalbumin (PV)-positive interneurons but not in pyramidal neurons. We found that the AMPAR auxiliary protein TARP γ-2 was specifically expressed in cortical interneurons. Erbin interacts with TARP γ-2 and is crucial for its stability. Deletion of the γ-2-interacting domain in Erbin attenuated surface AMPAR and excitatory transmission in PV-positive interneurons. Furthermore, we observed behavioral deficits in Erbin-null mice and in mice expressing an Erbin truncation mutant that is unable to interact with TARP γ-2. These observations demonstrate a crucial function for Erbin in AMPAR surface expression in cortical PV-positive interneurons and may contribute to a better understanding of psychiatric disorders.

HSP90 beta regulates rapsyn turnover and subsequent AChR cluster formation and maintenance.

Rapsyn, an acetylcholine receptor (AChR)-interacting protein, is essential for synapse formation at the neuromuscular junction (NMJ). Like many synaptic proteins, rapsyn turns over rapidly at synapses. However, little is known about molecular mechanisms that govern rapsyn stability. Using a differential mass-spectrometry approach, we identified heat-shock protein 90beta (HSP90beta) as a component in surface AChR clusters. The HSP90beta-AChR interaction required rapsyn and was stimulated by agrin. Inhibition of HSP90beta activity or expression, or disruption of its interaction with rapsyn attenuated agrin-induced formation of AChR clusters in vitro and impaired the development and maintenance of the NMJ in vivo. Finally, we showed that HSP90beta was necessary for rapsyn stabilization and regulated its proteasome-dependent degradation. Together, these results indicate a role of HSP90beta in NMJ development by regulating rapsyn turnover and subsequent AChR cluster formation and maintenance.

Bak regulates mitochondrial morphology and pathology during apoptosis by interacting with mitofusins.

Mitochondrial injury, characterized by outer membrane permeabilization and consequent release of apoptogenic factors, is a key to apoptosis of mammalian cells. Bax and Bak, two multidomain Bcl-2 family proteins, provide a requisite gateway to mitochondrial injury. However it is unclear how Bax and Bak cooperate to provoke mitochondrial injury and whether their roles are redundant. Here, we have identified a unique role of Bak in mitochondrial fragmentation, a seemingly morphological event that contributes to mitochondrial injury during apoptosis. We show that mitochondrial fragmentation is attenuated in Bak-deficient mouse embryonic fibroblasts, baby mouse kidney cells, and, importantly, also in primary neurons isolated from brain cortex of Bak-deficient mice. In sharp contrast, Bax deficiency does not prevent mitochondrial fragmentation during apoptosis. Bcl-2 and Bcl-XL inhibit mitochondrial fragmentation, and their inhibitory effects depend on the presence of Bak. Reconstitution of Bak into Bax/Bak double-knockout cells restores mitochondrial fragmentation, whereas reconstitution of Bax is much less effective. Bak interacts with Mfn1 and Mfn2, two mitochondrial fusion proteins. During apoptosis, Bak dissociates from Mfn2 and enhances the association with Mfn1. Mutation of Bak in the BH3 domain prevents its dissociation from Mfn2 and diminishes its mitochondrial fragmentation activity. This study has uncovered a previously unrecognized function of Bak in the regulation of mitochondrial morphological dynamics during apoptosis. By this function, Bak may collaborate with Bax to permeabilize the outer membrane of mitochondria, unleashing the apoptotic cascade.

Stimulated ErbB4 internalization is necessary for neuregulin signaling in neurons.

Neuregulin-1 (NRG1) plays an important role in neural development, synapse formation, and synaptic plasticity by activating ErbB receptor tyrosine kinases. Although ligand-induced endocytosis has been shown to be important for many receptor tyrosine kinases, whether NRG1 signaling depends on ErbB endocytosis remains controversial. Here, we provide evidence that ErbB4, a prominent ErbB protein in the brain, becomes internalized in NRG1-stimulated neurons. The induced ErbB4 endocytosis requires its kinase activity. Remarkably, inhibition of ErbB endocytosis attenuates NRG1-induced activation of Erk and Akt in neurons. These observations indicate a role of ErbB endocytosis in NRG1 signaling in neurons.

Neuronal transmission stimulates the phosphorylation of Kv1.4 channel at Ser229 through protein kinase A1.

Phosphorylation of voltage-gated K+ channels (Kv) is involved in regulation of neuronal excitability, synaptic plasticity and neuronal survival. Among Kv channels expressed in the CNS, Kv1.4 is located in the soma, dendrite and axon terminus of neurones in most regions of the brain. Here, we show that Ser229 found within the highly conserved T1 domain of Kv1.4 in cultured rat cortical neurones is phosphorylated by protein kinase A (PKA), as demonstrated by in vitro protein kinase assay and Western blotting with a polyclonal antibody specific against phosphorylated Ser229. Glutamate, high concentrations of K+ or K+ channel blockers known to increase neurotransmission all stimulated the phosphorylation of Kv1.4 at Ser229 via N-methyl-D-aspartate (NMDA), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor, whereas tetradotoxin (TTX), known to block neuronal transmission, and depletion of extracellular Ca2+ inhibited phosphorylation induced by tetraethylammonium (TEA), a non-selective K+ channel blocker. Mutation of Ser229 to Ala229 enhanced the current density. Taken together, elevation of the neuronal transmission stimulates the phosphorylation of Kv1.4 at Ser229 via the Ca2+ influx through NMDA receptor. Thus, it is possible that neuronal transmission regulates neuronal excitability partially through the phosphorylation of Kv1.4S229.