ER-PM Junctions on GABAergic Interneurons Are Organized by Neuregulin 2/VAP Interactions and Regulated by NMDA Receptors.

Neuregulins (NRGs) signal via ErbB receptors to regulate neural development, excitability, synaptic and network activity, and behaviors relevant to psychiatric disorders. Bidirectional signaling between NRG2/ErbB4 and NMDA receptors is thought to homeostatically regulate GABAergic interneurons in response to increased excitatory neurotransmission or elevated extracellular glutamate levels. Unprocessed proNRG2 forms discrete clusters on cell bodies and proximal dendrites that colocalize with the potassium channel Kv2.1 at specialized endoplasmic reticulum-plasma membrane (ER-PM) junctions, and NMDA receptor activation triggers rapid dissociation from ER-PM junctions and ectodomain shedding by ADAM10. Here, we elucidate the mechanistic basis of proNRG2 clustering at ER-PM junctions and its regulation by NMDA receptors. Importantly, we demonstrate that proNRG2 promotes the formation of ER-PM junctions by directly binding the ER-resident membrane tether VAP, like Kv2.1. The proNRG2 intracellular domain harbors two non-canonical, low-affinity sites that cooperatively mediate VAP binding. One of these is a cryptic and phosphorylation-dependent VAP binding motif that is dephosphorylated following NMDA receptor activation, thus revealing how excitatory neurotransmission promotes the dissociation of proNRG2 from ER-PM junctions. Therefore, proNRG2 and Kv2.1 can independently function as VAP-dependent organizers of neuronal ER-PM junctions. Based on these and prior studies, we propose that proNRG2 and Kv2.1 serve as co-regulated downstream effectors of NMDA receptors to homeostatically regulate GABAergic interneurons.

Pathway-specific contribution of parvalbumin interneuron NMDARs to synaptic currents and thalamocortical feedforward inhibition.

Prefrontal cortex (PFC) is a site of information convergence important for behaviors relevant to psychiatric disorders. Despite the importance of inhibitory GABAergic parvalbumin-expressing (PV+) interneurons to PFC circuit function and decades of interest in N-methyl-D-aspartate receptors (NMDARs) in these neurons, examples of defined circuit functions that depend on PV+ interneuron NMDARs have been elusive. Indeed, it remains controversial whether all PV+ interneurons contain functional NMDARs in adult PFC, which has major consequences for hypotheses of the pathogenesis of psychiatric disorders. Using a combination of fluorescent in situ hybridization, pathway-specific optogenetics, cell-type-specific gene ablation, and electrophysiological recordings from PV+ interneurons, here we resolve this controversy. We found that nearly 100% of PV+ interneurons in adult medial PFC (mPFC) express transcripts encoding GluN1 and GluN2B, and they have functional NMDARs. By optogenetically stimulating corticocortical and thalamocortical inputs to mPFC, we show that synaptic NMDAR contribution to PV+ interneuron EPSCs is pathway-specific, which likely explains earlier reports of PV+ interneurons without synaptic NMDAR currents. Lastly, we report a major contribution of NMDARs in PV+ interneurons to thalamus-mediated feedforward inhibition in adult mPFC circuits, suggesting molecular and circuit-based mechanisms for cognitive impairment under conditions of reduced NMDAR function. These findings represent an important conceptual advance that has major implications for hypotheses of the pathogenesis of psychiatric disorders.

Toxic and Phenotypic Effects of AAV_Cre Used to Transduce Mesencephalic Dopaminergic Neurons.

A popular approach to spatiotemporally target genes using the loxP/Cre recombination system is stereotaxic microinjection of adeno-associated virus (AAV) expressing Cre recombinase (AAV_Cre) in specific neuronal structures. Here, we report that AAV_Cre microinjection in the ventral tegmental area (VTA) of ErbB4 Cyt-1-floxed (ErbB4 Cyt-1fl/fl) mice at titers commonly used in the literature (~1012-1013 GC/mL) can have neurotoxic effects on dopaminergic neurons and elicit behavioral abnormalities. However, these effects of AAV_Cre microinjection are independent of ErbB4 Cyt-1 recombination because they are also observed in microinjected wild-type (WT) controls. Mice microinjected with AAV_Cre (1012-1013 GC/mL) exhibit reductions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) expression, loss of dopaminergic neurons, and they behaviorally become hyperactive, fail to habituate in the open field and exhibit sensorimotor gating deficits compared to controls microinjected with AAV_GFP. Importantly, these AAV_Cre non-specific effects are: (1) independent of serotype, (2) occur with vectors expressing either Cre or Cre-GFP fusion protein and (3) preventable by reducing viral titers by 1000-fold (1010 GC/mL), which retains sufficient recombination activity to target floxed genes. Our studies emphasize the importance of including AAV_Cre-injected WT controls in experiments because recombination-independent effects on gene expression, neurotoxicity and behaviors could be erroneously attributed to consequences of gene ablation.

Developmental, neurochemical, and behavioral analyses of ErbB4 Cyt-1 knockout mice.

Neuregulins (NRGs) and their cognate neuronal receptor ERBB4, which is expressed in GABAergic and dopaminergic neurons, regulate numerous behaviors in rodents and have been identified as schizophrenia at-risk genes. ErbB4 transcripts are alternatively spliced to generate isoforms that either include (Cyt-1) or exclude (Cyt-2) exon 26, which encodes a cytoplasmic domain that imparts ErbB4 receptors the ability to signal via the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Although ErbB4 Cyt-1/2 isoforms have been studied in transfected cultured cells, their functions in vivo remain unknown. Here, we generated ErbB4-floxed (ErbB4-Cyt1fl/fl ) mice to investigate the effects of germline (constitutive) and conditional (acute) deletions of the Cyt-1 exon. Overall receptor mRNA levels remain unchanged in germline ErbB4 Cyt-1 knockouts (Cyt-1 KOs), with all transcripts encoding Cyt-2 variants. In contrast to mice lacking all ErbB4 receptor function, GABAergic interneuron migration and number are unaltered in Cyt-1 KOs. However, basal extracellular dopamine (DA) levels in the medial prefrontal cortex are increased in Cyt-1 heterozygotes. Despite these neurochemical changes, Cyt-1 heterozygous and homozygous mice do not manifest behavioral abnormalities previously reported to be altered in ErbB4 null mice. To address the possibility that Cyt-2 variants compensate for the lack of Cyt-1 during development, we microinjected an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the DA-rich ventral tegmental area of adult ErbB4-Cyt1fl/fl mice to acutely target exon 26. These conditional Cyt-1 KOs were found to exhibit behavioral abnormalities in the elevated plus maze and startle response, consistent with the idea that late exon 26 ablations may circumvent compensation by Cyt-2 variants. Taken together, our observations indicate that ErbB4 Cyt-1 function in vivo is important for DA balance and behaviors in adults.

Transcytosis and trans-synaptic retention by postsynaptic ErbB4 underlie axonal accumulation of NRG3.

Neuregulins (NRGs) are EGF-like ligands associated with cognitive disorders. Unprocessed proNRG3 is cleaved by BACE1 to generate the mature membrane-bound NRG3 ligand, but the subcellular site of proNRG3 cleavage, mechanisms underlying its transport into axons, and presynaptic accumulation remain unknown. Using an optogenetic proNRG3 cleavage reporter (LA143-NRG3), we investigate the spatial-temporal dynamics of NRG3 processing and sorting in neurons. In dark conditions, unprocessed LA143-NRG3 is retained in the trans-Golgi network but, upon photoactivation, is cleaved by BACE1 and released from the TGN. Mature NRG3 then emerges on the somatodendritic plasma membrane from where it is re-endocytosed and anterogradely transported on Rab4+ vesicles into axons via transcytosis. By contrast, the BACE1 substrate APP is sorted into axons on Rab11+ vesicles. Lastly, by a mechanism we denote "trans-synaptic retention," NRG3 accumulates at presynaptic terminals by stable interaction with its receptor ErbB4 on postsynaptic GABAergic interneurons. We propose that trans-synaptic retention may account for polarized expression of other neuronal transmembrane ligands and receptors.

ErbB4 Null Mice Display Altered Mesocorticolimbic and Nigrostriatal Dopamine Levels as well as Deficits in Cognitive and Motivational Behaviors.

Natural genetic variants of Neuregulin1 (NRG1) and its cognate receptor ErbB4 are associated with a risk for schizophrenia. Whereas most studies on NRG1-ErbB4 signaling have focused on GABAergic interneurons, ErbB4 is also expressed by midbrain dopaminergic neurons where it modulates extracellular dopamine (DA) levels. Here, we report that extracellular steady-state levels of DA are reduced in the medial prefrontal cortex (mPFC; -65%), hippocampus (-53%) and nucleus accumbens (NAc; -35%), but are elevated in the dorsal striatum (+25%) of ErbB4 knock-out mice (ErbB4 KOs) relative to wild-type controls. This pattern of DA imbalance recapitulates the reported prefrontal cortical reduction and striatal increase of DA levels in schizophrenia patients. Next, we report on a battery of behavioral tasks used to evaluate locomotor, cognitive and motivational behaviors in ErbB4 KOs relative to controls. We found that ErbB4 KOs are hyperactive in a novel open field but not in their familiar home cage, are more sensitive to amphetamine, perform poorly in the T-maze and novel object recognition (NOR) tasks, exhibit reduced spatial learning and memory on the Barnes maze, and perform markedly worse in conditioned place preference (CPP) tasks when associating cued-reward palatable food with location. However, we found that the poor performance of ErbB4 KOs in CPP are likely due to deficits in spatial memory, instead of reward seeking, as ErbB4 KOs are more motivated to work for palatable food rewards. Our findings indicate that ErbB4 signaling affects tonic DA levels and modulates a wide array of behavioral deficits relevant to psychiatric disorders, including schizophrenia.

Correction to: NMDA Receptors Regulate Neuregulin 2 Binding to ER-PM Junctions and Ectodomain Release by ADAM10.

The original version of this article unfortunately contained an error in Title as the authors inadvertently deleted during revisions the last two words of the paper's title ("by ADAM10"), which is very important for explaining the article's content and impact.

NMDA Receptors Regulate Neuregulin 2 Binding to ER-PM Junctions and Ectodomain Release by ADAM10 [corrected].

Unprocessed pro-neuregulin 2 (pro-NRG2) accumulates on neuronal cell bodies at junctions between the endoplasmic reticulum and plasma membrane (ER-PM junctions). NMDA receptors (NMDARs) trigger NRG2 ectodomain shedding from these sites followed by activation of ErbB4 receptor tyrosine kinases, and ErbB4 signaling cell-autonomously downregulates intrinsic excitability of GABAergic interneurons by reducing voltage-gated sodium channel currents. NMDARs also promote dispersal of Kv2.1 clusters from ER-PM junctions and cause a hyperpolarizing shift in its voltage-dependent channel activation, suggesting that NRG2/ErbB4 and Kv2.1 work together to regulate intrinsic interneuron excitability in an activity-dependent manner. Here we explored the cellular processes underlying NMDAR-dependent NRG2 shedding in cultured rat hippocampal neurons. We report that NMDARs control shedding by two separate but converging mechanisms. First, NMDA treatment disrupts binding of pro-NRG2 to ER-PM junctions by post-translationally modifying conserved Ser/Thr residues in its intracellular domain. Second, using a mutant NRG2 protein that cannot be modified at these residues and that fails to accumulate at ER-PM junctions, we demonstrate that NMDARs also directly promote NRG2 shedding by ADAM-type metalloproteinases. Using pharmacological and shRNA-mediated knockdown, and metalloproteinase overexpression, we unexpectedly find that ADAM10, but not ADAM17/TACE, is the major NRG2 sheddase acting downstream of NMDAR activation. Together, these findings reveal how NMDARs exert tight control over the NRG2/ErbB4 signaling pathway, and suggest that NRG2 and Kv2.1 are co-regulated components of a shared pathway that responds to elevated extracellular glutamate levels.

Detection and Quantification of Multiple RNA Sequences Using Emerging Ultrasensitive Fluorescent In Situ Hybridization Techniques.

Fluorescent detection of transcripts using RNAscope has quickly become a standard in situ hybridization (ISH) approach in neuroscience with over 400 publications since its introduction in 2012. RNAscope's sensitivity and specificity allow the simultaneously detection of up to three low abundance mRNAs in single cells (i.e., multiplexing) and, in contrast to other ISH techniques, RNAscope is performed in 1 day. BaseScope, a newer ultrasensitive platform, uses improved amplification chemistry of single oligonucleotide probe pairs (∼50 bases). This technique allows discrimination of single nucleotide polymorphisms or splice variants that differ by short exons. A present limitation of BaseScope is that expression analysis is limited to a single gene (i.e., single-plexing). This article outlines detailed protocols for both RNAscope and BaseScope in neuronal tissue. We discuss how to perform ISH experiments using either fresh-frozen or formalin-fixed paraffin-embedded sections, as well as dissociated cultured neurons. We also outline how to obtain quantitative data from hybridized tissue sections. © 2019 by John Wiley & Sons, Inc.

The locus coeruleus drives disinhibition in the midline thalamus via a dopaminergic mechanism.

The paraventricular nucleus of the thalamus (PVT) is increasingly being recognized as a critical node linking stress detection to the emergence of adaptive behavioral responses to stress. However, despite growing evidence implicating the PVT in stress processing, the neural mechanisms by which stress impacts PVT neurocircuitry and promotes stressed states remain unknown. Here we show that stress exposure drives a rapid and persistent reduction of inhibitory transmission onto projection neurons of the posterior PVT (pPVT). This stress-induced disinhibition of the pPVT was associated with a locus coeruleus-mediated rise in the extracellular concentration of dopamine in the midline thalamus, required the function of dopamine D2 receptors on PVT neurons, and increased sensitivity to stress. Our findings define the locus coeruleus as an important modulator of PVT function: by controlling the inhibitory tone of the pPVT, it modulates the excitability of pPVT projection neurons and controls stress responsivity.

Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome.

The hereditary spastic paraplegias (HSP) are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in subunits of the heterotetrameric (ε-ß4-µ4-σ4) adaptor protein 4 (AP-4) complex cause an autosomal recessive form of complicated HSP referred to as "AP-4 deficiency syndrome". In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. The pathogenetic mechanism, however, remains poorly understood. Here we report the characterization of a knockout (KO) mouse for the AP4E1 gene encoding the ε subunit of AP-4. We find that AP-4 ε KO mice exhibit a range of neurological phenotypes, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, AP-4 ε KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses show that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in skin fibroblasts from patients with mutations in the µ4 subunit of AP-4 and in various neuronal types in AP-4 ε KO mice. ATG9A mislocalization is associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 ε KO neurons. These findings indicate that the AP-4 ε KO mouse is a suitable animal model for AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder.

A histone deacetylase 3-dependent pathway delimits peripheral myelin growth and functional regeneration.

Deficits in Schwann cell-mediated remyelination impair functional restoration after nerve damage, contributing to peripheral neuropathies. The mechanisms mediating block of remyelination remain elusive. Here, through small-molecule screening focusing on epigenetic modulators, we identified histone deacetylase 3 (HDAC3; a histone-modifying enzyme) as a potent inhibitor of peripheral myelinogenesis. Inhibition of HDAC3 enhanced myelin growth and regeneration and improved functional recovery after peripheral nerve injury in mice. HDAC3 antagonizes the myelinogenic neuregulin-PI3K-AKT signaling axis. Moreover, genome-wide profiling analyses revealed that HDAC3 represses promyelinating programs through epigenetic silencing while coordinating with p300 histone acetyltransferase to activate myelination-inhibitory programs that include the HIPPO signaling effector TEAD4 to inhibit myelin growth. Schwann cell-specific deletion of either Hdac3 or Tead4 in mice resulted in an elevation of myelin thickness in sciatic nerves. Thus, our findings identify the HDAC3-TEAD4 network as a dual-function switch of cell-intrinsic inhibitory machinery that counters myelinogenic signals and maintains peripheral myelin homeostasis, highlighting the therapeutic potential of transient HDAC3 inhibition for improving peripheral myelin repair.

A Novel Ultrasensitive In Situ Hybridization Approach to Detect Short Sequences and Splice Variants with Cellular Resolution.

Investigating the expression of RNAs that differ by short or single nucleotide sequences at a single-cell level in tissue has been limited by the sensitivity and specificity of in situ hybridization (ISH) techniques. Detection of short isoform-specific sequences requires RNA isolation for PCR analysis-an approach that loses the regional and cell-type-specific distribution of isoforms. Having the capability to distinguish the differential expression of RNA variants in tissue is critical because alterations in mRNA splicing and editing, as well as coding single nucleotide polymorphisms, have been associated with numerous cancers, neurological and psychiatric disorders. Here we introduce a novel highly sensitive single-probe colorimetric/fluorescent ISH approach that targets short exon/exon RNA splice junctions using single-pair oligonucleotide probes (~ 50 bp). We use this approach to investigate, with single-cell resolution, the expression of four transcripts encoding the neuregulin (NRG) receptor ErbB4 that differ by alternative splicing of exons encoding two juxtamembrane (JMa/JMb) and two cytoplasmic (CYT-1/CYT-2) domains that alter receptor stability and signaling modes, respectively. By comparing ErbB4 hybridization on sections from wild-type and ErbB4 knockout mice (missing exon 2), we initially demonstrate that single-pair probes provide the sensitivity and specificity to visualize and quantify the differential expression of ErbB4 isoforms. Using cell-type-specific GFP reporter mice, we go on to demonstrate that expression of ErbB4 isoforms differs between neurons and oligodendrocytes, and that this differential expression of ErbB4 isoforms is evolutionarily conserved to humans. This single-pair probe ISH approach, known as BaseScope, could serve as an invaluable diagnostic tool to detect alternative spliced isoforms, and potentially single base polymorphisms, associated with disease.

Neuronal correlates of ketamine and walking induced gamma oscillations in the medial prefrontal cortex and mediodorsal thalamus.

Alterations in the function of the medial prefrontal cortex (mPFC) and its major thalamic source of innervation, the mediodorsal (MD) thalamus, have been hypothesized to contribute to the symptoms of schizophrenia. The NMDAR antagonist ketamine, used to model schizophrenia, elicits a brain state resembling early stage schizophrenia characterized by cognitive deficits and increases in cortical low gamma (40-70 Hz) power. Here we sought to determine how ketamine differentially affects spiking and gamma local field potential (LFP) activity in the rat mPFC and MD thalamus. Additionally, we investigated the ability of drugs targeting the dopamine D4 receptor (D4R) to modify the effects of ketamine on gamma activity as a measure of potential cognitive therapeutic efficacy. Rats were trained to walk on a treadmill to reduce confounds related to hyperactivity after ketamine administration (10 mg/kg s.c.) while recordings were obtained from electrodes chronically implanted in the mPFC and MD thalamus. Ketamine increased gamma LFP power in mPFC and MD thalamus in a similar frequency range, yet did not increase thalamocortical synchronization. Ketamine also increased firing rates and spike synchronization to gamma oscillations in the mPFC but decreased both measures in MD thalamus. Conversely, walking alone increased both firing rates and spike-gamma LFP correlations in both mPFC and MD thalamus. The D4R antagonist alone (L-745,870) had no effect on gamma LFP power during treadmill walking, although it reversed increases induced by the D4R agonist (A-412997) in both mPFC and MD thalamus. Neither drug altered ketamine-induced changes in gamma power or firing rates in the mPFC. However, in MD thalamus, the D4R agonist increased ketamine-induced gamma power and prevented ketamine's inhibitory effect on firing rates. Results provide new evidence that ketamine differentially modulates spiking and gamma power in MD thalamus and mPFC, supporting a potential role for both areas in contributing to ketamine-induced schizophrenia-like symptoms.

Structural Similarities between Neuregulin 1-3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons.

The Neuregulin (NRG) family of ErbB ligands is comprised of numerous variants originating from the use of different genes, alternative promoters, and splice variants. NRGs have generally been thought to be transported to axons and presynaptic terminals where they signal via ErbB3/4 receptors in paracrine or juxtacrine mode. However, we recently demonstrated that unprocessed pro-NRG2 accumulates on cell bodies and proximal dendrites, and that NMDAR activity is required for shedding of its ectodomain by metalloproteinases. Here we systematically investigated the subcellular distribution and processing of major NRG isoforms in rat hippocampal neurons. We show that NRG1 isotypes I and II, which like NRG2 are single-pass transmembrane proteins with an Ig-like domain, share the same subcellular distribution and ectodomain shedding properties. We furthermore show that NRG3, like CRD-NRG1, is a dual-pass transmembrane protein that harbors a second transmembrane domain near its amino terminus. Both NRG3 and CRD-NRG1 cluster on axons through juxtacrine interactions with ErbB4 present on GABAergic interneurons. Interestingly, although single-pass NRGs accumulate as unprocessed proforms, axonal puncta of CRD-NRG1 and NRG3 are comprised of processed protein. Mutations of CRD-NRG1 and NRG3 that render them resistant to BACE cleavage, as well as BACE inhibition, result in the loss of axonal puncta and in the accumulation of unprocessed proforms in neuronal soma. Together, these results define two groups of NRGs with distinct membrane topologies and fundamentally different targeting and processing properties in central neurons. The implications of this functional diversity for the regulation of neuronal processes by the NRG/ErbB pathway are discussed.SIGNIFICANCE STATEMENT Numerous Neuregulins (NRGs) are generated through the use of different genes, promoters, and alternative splicing, but the functional significance of this evolutionary conserved diversity remains poorly understood. Here we show that NRGs can be categorized by their membrane topologies. Single-pass NRGs, such as NRG1 Types I/II and NRG2, accumulate as unprocessed proforms on cell bodies, and their ectodomains are shed by metalloproteinases in response to NMDA receptor activation. By contrast, dual-pass CRD-NRG1 and NRG3 are constitutively processed by BACE and accumulate on axons where they interact with ErbB4 in juxtacrine mode. These findings reveal a previously unknown functional relationship between membrane topology, protein processing, and subcellular distribution, and suggest that single- and dual-pass NRGs regulate neuronal functions in fundamentally different ways.

A negative feedback loop controls NMDA receptor function in cortical interneurons via neuregulin 2/ErbB4 signalling.

The neuregulin receptor ErbB4 is an important modulator of GABAergic interneurons and neural network synchronization. However, little is known about the endogenous ligands that engage ErbB4, the neural processes that activate them or their direct downstream targets. Here we demonstrate, in cultured neurons and in acute slices, that the NMDA receptor is both effector and target of neuregulin 2 (NRG2)/ErbB4 signalling in cortical interneurons. Interneurons co-express ErbB4 and NRG2, and pro-NRG2 accumulates on cell bodies atop subsurface cisternae. NMDA receptor activation rapidly triggers shedding of the signalling-competent NRG2 extracellular domain. In turn, NRG2 promotes ErbB4 association with GluN2B-containing NMDA receptors, followed by rapid internalization of surface receptors and potent downregulation of NMDA but not AMPA receptor currents. These effects occur selectively in ErbB4-positive interneurons and not in ErbB4-negative pyramidal neurons. Our findings reveal an intimate reciprocal relationship between ErbB4 and NMDA receptors with possible implications for the modulation of cortical microcircuits associated with cognitive deficits in psychiatric disorders.

Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells.

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.

Disruption of the ErbB signaling in adolescence increases striatal dopamine levels and affects learning and hedonic-like behavior in the adult mouse.

The ErbB signaling pathway has been genetically and functionally implicated in schizophrenia. Numerous findings support the dysregulation of Neuregulin (NRG) and epidermal growth factor (EGF) signaling in schizophrenia. However, it is unclear whether alterations of these pathways in the adult brain or during development are involved in the pathophysiology of schizophrenia. Herein we characterized the behavioral profile and molecular changes resulting from pharmacologically blocking the ErbB signaling pathway during a critical period in the development of decision making, planning, judgments, emotions, social cognition and cognitive skills, namely adolescence. We demonstrate that chronic administration of the pan-ErbB kinase inhibitor JNJ-28871063 (JNJ) to adolescent mice elevated striatal dopamine levels and reduced preference for sucrose without affecting locomotor activity and exploratory behavior. In adulthood, adolescent JNJ-treated mice continue to consume less sucrose and needed significantly more correct-response trials to reach the learning criterion during the discrimination phase of the T-maze reversal learning task than their saline-injected controls. In addition, JNJ mice exhibited deficit in reference memory but not in working memory as measured in the radial arm maze. Inhibition of the pathway during adolescence did not affect exploratory behavior and locomotor activity in the open field, social interaction, social memory, and reversal learning in adult mice. Our data suggest that alteration of ErbB signaling during adolescence resulted in changes in the dopaminergic systems that emerge in pathological learning and hedonic behavior in adulthood, and pinpoints the possible role of the pathway in the development of cognitive skills and motivated behavior.

Detection of differential fetal and adult expression of chloride intracellular channel 4 (CLIC4) protein by analysis of a green fluorescent protein knock-in mouse line.

BACKGROUND: Chloride Intracellular Channel 4 (CLIC4) is one of seven members in the closely related CLIC protein family. CLIC4 is involved in multiple cellular processes including apoptosis, cellular differentiation, inflammation and endothelial tubulogenesis. Despite over a decade of research, no comprehensive in situ expression analysis of CLIC4 in a living organism has been reported. In order to fulfill this goal, we generated a knock-in mouse to express Green Fluorescent Protein (GFP) from the CLIC4 locus, thus substituting the GFP coding region for CLIC4. We used GFP protein expression to eliminate cross reaction with other CLIC family members. RESULTS: We analyzed CLIC4 expression during embryonic development and adult organs. During mid and late gestation, CLIC4 expression is modulated particularly in fetal brain, heart, thymus, liver and kidney as well as in developing brown adipose tissue and stratifying epidermis. In the adult mouse, CLIC4 is highly expressed globally in vascular endothelial cells as well as in liver, lung alveolar septae, pancreatic acini, spermatogonia, renal proximal tubules, cardiomyocytes and thymic epithelial cells. Neural expression included axonal tracks, olfactory bulb, Purkinje cell layer and dentate gyrus. Renal CLIC4 expression was most pronounced in proximal tubules, although altered renal function was not detected in the absence of CLIC4. Myeloid cells and B cells of the spleen are rich in CLIC4 expression as are CD4 and CD8 positive T cells. CONCLUSIONS: In a comprehensive study detailing CLIC4 expression in situ in a mouse model that excludes cross reaction with other family members, we were able to document previously unreported expression for CLIC4 in developing fetus, particularly the brain. In addition, compartmentalized expression of CLIC4 in specific adult tissues and cells provides a focus to explore potential functions of this protein not addressed previously.

ErbB4 reduces synaptic GABAA currents independent of its receptor tyrosine kinase activity.

ErbB4 signaling in the central nervous system is implicated in neuropsychiatric disorders and epilepsy. In cortical tissue, ErbB4 associates with excitatory synapses located on inhibitory interneurons. However, biochemical and histological data described herein demonstrate that the vast majority of ErbB4 is extrasynaptic and detergent-soluble. To explore the function of this receptor population, we used unbiased proteomics, in combination with electrophysiological, biochemical, and cell biological techniques, to identify a clinically relevant ErbB4-interacting protein, the GABAA receptor α1 subunit (GABAR α1). We show that ErbB4 and GABAR α1 are robustly coexpressed in hippocampal interneurons, and that ErbB4-null mice have diminished cortical GABAR α1 expression. Moreover, we characterize a Neuregulin-mediated ErbB4 signaling modality, independent of receptor tyrosine kinase activity, that couples ErbB4 to decreased postsynaptic GABAR currents on inhibitory interneurons. Consistent with an evolving understanding of GABAR trafficking, this pathway requires both clathrin-mediated endocytosis and protein kinase C to reduce GABAR inhibitory currents, surface GABAR α1 expression, and colocalization with the inhibitory postsynaptic protein gephyrin. Our results reveal a function of ErbB4, independent of its tyrosine kinase activity, that modulates postsynaptic inhibitory control of hippocampal interneurons and may provide a novel pharmacological target in the treatment of neuropsychiatric disorders and epilepsy.