The TAM Subfamily of Receptor Tyrosine Kinases: The Early Years.

The TAMs are a subfamily of receptor tyrosine kinases (RTKs) comprised of three members, Tyro3, Axl and Mer. Evidence in support of the existence of this subfamily emerged from a screen for novel RTKs performed in the laboratory of Dr. Greg Lemke in 1991. A PCR-based approach to selectively amplify tyrosine kinase-specific genes yielded 27 different tyrosine kinase genes, of which 13 were novel (the "Tyros"). Of these, Tyro3, 7 and 12 were more closely related to each other than to any other kinases and it was proposed that they constituted a novel subfamily of RTKs. Additional support for this hypothesis required determining the complete sequences for these receptor tyrosine kinases. By the end of 1991, full-length sequences for Tyro7 (Axl) revealed a unique extracellular domain organization that included two immunoglobulin-like domains and two fibronectin type III repeats. In 1994, the complete sequences for Tyro12 (Mer) and Tyro3 were shown to have an extracellular region domain structure similar to that of Axl. In 1995, Gas6 and Pros1 were reported as ligands for Tyro3 and Axl, setting the stage for functional studies. The Lemke lab and its many trainees have since played leading roles in elucidating the physiological relevance of the TAMs.

Developmental Disruption of Erbb4 in Pet1+ Neurons Impairs Serotonergic Sub-System Connectivity and Memory Formation.

The serotonergic system of mammals innervates virtually all the central nervous system and regulates a broad spectrum of behavioral and physiological functions. In mammals, serotonergic neurons located in the rostral raphe nuclei encompass diverse sub-systems characterized by specific circuitry and functional features. Substantial evidence suggest that functional diversity of serotonergic circuits has a molecular and connectivity basis. However, the landscape of intrinsic developmental mechanisms guiding the formation of serotonergic sub-systems is unclear. Here, we employed developmental disruption of gene expression specific to serotonergic subsets to probe the contribution of the tyrosine kinase receptor ErbB4 to serotonergic circuit formation and function. Through an in vivo loss-of-function approach, we found that ErbB4 expression occurring in a subset of serotonergic neurons, is necessary for axonal arborization of defined long-range projections to the forebrain but is dispensable for the innervation of other targets of the serotonergic system. We also found that Erbb4-deletion does not change the global excitability or the number of neurons with serotonin content in the dorsal raphe nuclei. In addition, ErbB4-deficiency in serotonergic neurons leads to specific behavioral deficits in memory processing that involve aversive or social components. Altogether, our work unveils a developmental mechanism intrinsically acting through ErbB4 in subsets of serotonergic neurons to orchestrate a precise long-range circuit and ultimately involved in the formation of emotional and social memories.

Neuregulin signaling mediates the acute and sustained antidepressant effects of subanesthetic ketamine.

Subanesthetic ketamine evokes rapid antidepressant effects in human patients that persist long past ketamine's chemical half-life of ~2 h. Ketamine's sustained antidepressant action may be due to modulation of cortical plasticity. We find that ketamine ameliorates depression-like behavior in the forced swim test in adult mice, and this depends on parvalbumin-expressing (PV) neuron-directed neuregulin-1 (NRG1)/ErbB4 signaling. Ketamine rapidly downregulates NRG1 expression in PV inhibitory neurons in mouse medial prefrontal cortex (mPFC) following a single low-dose ketamine treatment. This NRG1 downregulation in PV neurons co-tracks with the decreases in synaptic inhibition to mPFC excitatory neurons for up to a week. This results from reduced synaptic excitation to PV neurons, and is blocked by exogenous NRG1 as well as by PV targeted ErbB4 receptor knockout. Thus, we conceptualize that ketamine's effects are mediated through rapid and sustained cortical disinhibition via PV-specific NRG1 signaling. Our findings reveal a novel neural plasticity-based mechanism for ketamine's acute and long-lasting antidepressant effects.

Subanesthetic Ketamine Reactivates Adult Cortical Plasticity to Restore Vision from Amblyopia.

Subanesthetic ketamine evokes rapid and long-lasting antidepressant effects in human patients. The mechanism for ketamine's effects remains elusive, but ketamine may broadly modulate brain plasticity processes. We show that single-dose ketamine reactivates adult mouse visual cortical plasticity and promotes functional recovery of visual acuity defects from amblyopia. Ketamine specifically induces downregulation of neuregulin-1 (NRG1) expression in parvalbumin-expressing (PV) inhibitory neurons in mouse visual cortex. NRG1 downregulation in PV neurons co-tracks both the fast onset and sustained decreases in synaptic inhibition to excitatory neurons, along with reduced synaptic excitation to PV neurons in vitro and in vivo following a single ketamine treatment. These effects are blocked by exogenous NRG1 as well as PV targeted receptor knockout. Thus, ketamine reactivation of adult visual cortical plasticity is mediated through rapid and sustained cortical disinhibition via downregulation of PV-specific NRG1 signaling. Our findings reveal the neural plasticity-based mechanism for ketamine-mediated functional recovery from adult amblyopia.

Neuregulins 1, 2, and 3 Promote Early Neurite Outgrowth in ErbB4-Expressing Cortical GABAergic Interneurons.

The neuregulins (Nrgs 1-4) are a family of signaling molecules that play diverse roles in the nervous system. Nrg1 has been implicated in the formation of synapses and in synaptic plasticity. Previous studies have shown Nrg1 can affect neurite outgrowth in several neuronal populations, while the role of Nrg2 and Nrg3 in this process has remained understudied. The Nrgs can bind and activate the ErbB4 receptor tyrosine kinase which is preferentially expressed in GABAergic interneurons in the rodent hippocampus and cerebral cortex. In the present study, we evaluated the effects of Nrgs 1, 2, and 3 on neurite outgrowth of dissociated rat cortical ErbB4-positive (+)/GABA+ interneurons in vitro. All three Nrgs were able to promote neurite outgrowth during the first 2 days in vitro, with increases detected for both the axon (116-120%) and other neurites (100-120%). Increases in the average number of primary and secondary neurites were also observed. Treatment with the Nrgs for an additional 3 days promoted an increase in axonal length (86-96%), with only minimal effects on the remaining neurites (8-13%). ErbB4 expression persisted throughout the dendritic arbor and cell soma at all stages examined, while its expression in the axon was transient and declined with cell maturation. ErbB4 overexpression in GABAergic neurons promoted neurite outgrowth, an effect that was potentiated by Nrg treatment. These results show that Nrgs 1, 2, and 3 are each capable of influencing dendritic and axonal growth at early developmental stages in GABAergic neurons grown in vitro.

Neuregulin and ErbB expression is regulated by development and sensory experience in mouse visual cortex.

Neuregulins (NRGs) are protein ligands that impact neural development and circuit function. NRGs signal through the ErbB receptor tyrosine kinase family. NRG1/ErbB4 signaling in parvalbumin-expressing (PV) inhibitory interneurons is critical for visual cortical plasticity. There are multiple types of NRGs and ErbBs that can potentially contribute to visual cortical plasticity at different developmental stages. Thus, it is important to understand the normal developmental expression profiles of NRGs and ErbBs in specific neuron types in the visual cortex, and to study whether and how their expression changes in PV inhibitory neurons and excitatory neurons track with sensory perturbation. Cell type-specific translating ribosome affinity purification and qPCR was used to compare mRNA expression of nrg1,2,3,4 and erbB1,2,3,4 in PV and excitatory neurons in mouse visual cortex. We show that the expression of nrg1 and nrg3 decreases in PV neurons at the critical period peak, postnatal day 28 (P28) after monocular deprivation and dark rearing, and in the adult cortex (at P104) after 2-week long dark exposure. In contrast, nrg1 expression by excitatory neurons is unchanged at P28 and P104 following sensory deprivation, whereas nrg3 expression by excitatory neurons shows changes depending on the age and the mode of sensory deprivation. ErbB4 expression in PV neurons remains consistently high and does not appear to change in response to sensory deprivation. These data provide new important details of cell type-specific NRG/ErbB expression in the visual cortex and support that NRG1/ErbB4 signaling is implicated in both critical period and adult visual cortical plasticity.

Developmental expression of Neuregulin-3 in the rat central nervous system.

Neuregulin-3 (Nrg3) is a member of the Nrg family of growth factors identified as risk factors for schizophrenia. There are three Nrgs expressed in the nervous system (Nrg1-3) and of these Nrg1 has been the best characterized. To set the groundwork for elucidating neural roles for Nrg3, we studied its expression in the rat brain at both the RNA and protein levels. Using an antibody developed against Nrg3, we observed a developmental increase of Nrg3 protein expression from embryonic stages to adulthood and determined that it carries O-linked carbohydrates. In cortical neuronal cultures, transfected Neuro2a cells, and brain tissue sections Nrg3 protein was localized to the soma, neurites, and to the Golgi apparatus, where it is prominently expressed. Nrg3 was detected in excitatory, GABAergic and parvalbumin-expressing inhibitory neurons while expression in glia was limited. Nrg3 mRNA and protein were widely expressed during both embryonic and postnatal ages. At E17, Nrg3 was detected within the cortical plate and ventricular zone suggesting possible roles in cell proliferation or migration. At postnatal ages, Nrg3 was abundantly expressed throughout the cerebral cortex and hippocampus. Multiple thalamic nuclei expressed Nrg3, while detection in the striatum was limited. In the cerebellum, Nrg3 was found in both Purkinje cells and granule neurons. In the rodent brain, Nrg3 is the most abundantly expressed of the Nrgs and its patterns of expression differ both temporally and spatially from that of Nrg1 and Nrg2. These findings suggest that Nrg3 plays roles that are distinct from the other Nrg family members.

Neuregulin-1/ErbB4 Signaling Regulates Visual Cortical Plasticity.

Experience alters cortical networks through neural plasticity mechanisms. During a developmental critical period, the most dramatic consequence of occluding vision through one eye (monocular deprivation) is a rapid loss of excitatory synaptic inputs to parvalbumin-expressing (PV) inhibitory neurons in visual cortex. Subsequent cortical disinhibition by reduced PV cell activity allows for excitatory ocular dominance plasticity. However, the molecular mechanisms underlying critical period synaptic plasticity are unclear. Here we show that brief monocular deprivation during the critical period downregulates neuregulin-1(NRG1)/ErbB4 signaling in PV neurons, causing retraction of excitatory inputs to PV neurons. Exogenous NRG1 rapidly restores excitatory inputs onto deprived PV cells through downstream PKC-dependent activation and AMPA receptor exocytosis, thus enhancing PV neuronal inhibition to excitatory neurons. NRG1 treatment prevents the loss of deprived eye visual cortical responsiveness in vivo. Our findings reveal molecular, cellular, and circuit mechanisms of NRG1/ErbB4 in regulating the initiation of critical period visual cortical plasticity.

Interneuronal DISC1 regulates NRG1-ErbB4 signalling and excitatory-inhibitory synapse formation in the mature cortex.

Neuregulin-1 (NRG1) and its receptor ErbB4 influence several processes of neurodevelopment, but the mechanisms regulating this signalling in the mature brain are not well known. DISC1 is a multifunctional scaffold protein that mediates many cellular processes. Here we present a functional relationship between DISC1 and NRG1-ErbB4 signalling in mature cortical interneurons. By cell type-specific gene modulation in vitro and in vivo including in a mutant DISC1 mouse model, we demonstrate that DISC1 inhibits NRG1-induced ErbB4 activation and signalling. This effect is likely mediated by competitive inhibition of binding of ErbB4 to PSD95. Finally, we show that interneuronal DISC1 affects NRG1-ErbB4-mediated phenotypes in the fast spiking interneuron-pyramidal neuron circuit. Post-mortem brain analyses and some genetic studies have reported interneuronal deficits and involvement of the DISC1, NRG1 and ErbB4 genes in schizophrenia, respectively. Our results suggest a mechanism by which cross-talk between DISC1 and NRG1-ErbB4 signalling may contribute to these deficits.

Cross-generational impact of a male murine pheromone 2-sec-butyl-4,5- dihydrothiazole in female mice.

The current understanding of the activity of mammalian pheromones is that endocrine and behavioural effects are limited to the exposed individuals. Here, we demonstrate that the nasal exposure of female mice to a male murine pheromone stimulates expansion of mammary glands, leading to prolonged nursing of pups. Subsequent behavioural testing of the pups from pheromone-exposed dams exhibited enhanced learning. Sialic acid components in the milk are known to be involved in brain development. We hypothesized that the offspring might have received more of this key nutrient that promotes brain development. The mRNA for polysialyltransferase, which produces polysialylated neural cell adhesion molecules related to brain development,was increased in the brain of offspring of pheromone-exposed dams at post-natal day 10, while it was not different at embryonic stages, indicating possible differential brain development during early post-natal life.

Homologous recombination using bacterial artificial chromosomes.

This protocol introduces the technique of homologous recombination in bacteria to insert a linear DNA fragment into bacterial artificial chromosomes (BACs). Homologous recombination allows the modification of large DNA molecules, in contrast with conventional restriction endonuclease-based strategies, which cleave large DNAs into numerous fragments and are unlikely to permit the precise targeting afforded by recombination-based approaches. The method uses a phage lambda-derived recombination system (using exo, beta, and gam) as well as other enzymatic activities provided by the host (Escherichia coli). In the method described here, a DNA fragment encoding enhanced cyan fluorescent protein is inserted immediately after the start codon of the gene encoding choline acetyltransferase ("ChAT"), the final enzyme in acetylcholine biosynthesis, using homologous recombination between sequences that are present both on the introduced DNA fragment and in the target BAC. The desired recombination products are identified via positive selection for resistance to kanamycin. In principle, the resulting modified BAC could be used to produce transgenic mice that express this fluorescent protein in cholinergic neurons. The approach described here could be used to insert any DNA fragment.

ErbB4 regulation of a thalamic reticular nucleus circuit for sensory selection.

Selective processing of behaviorally relevant sensory inputs against irrelevant ones is a fundamental cognitive function whose impairment has been implicated in major psychiatric disorders. It is known that the thalamic reticular nucleus (TRN) gates sensory information en route to the cortex, but the underlying mechanisms remain unclear. Here we show in mice that deficiency of the Erbb4 gene in somatostatin-expressing TRN neurons markedly alters behaviors that are dependent on sensory selection. Whereas the performance of the Erbb4-deficient mice in identifying targets from distractors was improved, their ability to switch attention between conflicting sensory cues was impaired. These behavioral changes were mediated by an enhanced cortical drive onto the TRN that promotes the TRN-mediated cortical feedback inhibition of thalamic neurons. Our results uncover a previously unknown role of ErbB4 in regulating cortico-TRN-thalamic circuit function. We propose that ErbB4 sets the sensitivity of the TRN to cortical inputs at levels that can support sensory selection while allowing behavioral flexibility.

Pheromone-induced cell proliferation in the murine subventricular zone.

Enhancement of adult neurogenesis in female mice was previously demonstrated through exposure to soiled bedding from males, although the identity of relevant chemosignals has remained unknown. The farnesenes and SBT (2-sec-butyl-4,5-dihydrothiazole) are male murine pheromones that dominant males secrete at higher levels. Previous studies have shown that they induce oestrus in female mice. We have recently shown that these pheromones strongly increase cell proliferation in the SVZ (subventricular zone) of adult female mice. In addition, we found that a female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. 2,5-dimethylpyrazine is a female pheromone that is secreted when females are housed in large groups and it was originally found to suppress oestrus in females. We found that it does not have suppressive effect on the cell proliferation in the SVZ of females. Similarly, male murine pheromones, SBT and the farnesenes, do not show a suppressive effect on the cell proliferation in the SVZ of males. Our results demonstrated that pheromonal communication between males and females has strong stimulatory effect on both the reproductive physiology and brain cell proliferation, but intrasex pheromonal exchanges do not reduce progenitor proliferation in these brain regions.

Stimulation of cell proliferation in the subventricular zone by synthetic murine pheromones.

Adult neurogenesis in female mice is known to be enhanced by exposure to soiled bedding from males, although the identity of the relevant chemosignals has remained unknown. Here we show that the previously recognized male murine pheromones, the farnesenes and 2-sec-butyl-4,5-dihydrothiazole (SBT), strongly increase cell proliferation in the subventricular zone (SVZ) of adult female mice, but not younger female mice. In addition, we found that a unique female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. SBT stimulated cell proliferation in the SVZ of only adult females and not in young adult or pre- and post-puberty females. Our study suggests that pheromonal communication between males and females is enhancing reproductive success by controlling the estrous cycle and by promoting cell proliferation in a reciprocal manner.

Arl13b in primary cilia regulates the migration and placement of interneurons in the developing cerebral cortex.

Coordinated migration and placement of interneurons and projection neurons lead to functional connectivity in the cerebral cortex; defective neuronal migration and the resultant connectivity changes underlie the cognitive defects in a spectrum of neurological disorders. Here we show that primary cilia play a guiding role in the migration and placement of postmitotic interneurons in the developing cerebral cortex and that this process requires the ciliary protein, Arl13b. Through live imaging of interneuronal cilia, we show that migrating interneurons display highly dynamic primary cilia and we correlate cilia dynamics with the interneuron's migratory state. We demonstrate that the guidance cue receptors essential for interneuronal migration localize to interneuronal primary cilia, but their concentration and dynamics are altered in the absence of Arl13b. Expression of Arl13b variants known to cause Joubert syndrome induce defective interneuronal migration, suggesting that defects in cilia-dependent interneuron migration may in part underlie the neurological defects in Joubert syndrome patients.

Cross-phosphorylation, signaling and proliferative functions of the Tyro3 and Axl receptors in Rat2 cells.

The dysregulation of receptor protein tyrosine kinase (RPTK) function can result in changes in cell proliferation, cell growth and metastasis leading to malignant transformation. Among RPTKs, the TAM receptor family composed of three members Tyro3, Axl, and Mer has been recognized to have a prominent role in cell transformation. In this study we analyzed the consequences of Tyro3 overexpression on cell proliferation, activation of signaling pathways and its functional interactions with Axl. Overexpression of Tyro3 in the Rat2 cell line that expresses Axl, but not Mer or Tyro3, resulted in a 5 fold increase in cell proliferation. This increase was partially blocked by inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway but not by inhibitors of the phosphatidylinositol 3-kinase (PI(3)K) signaling pathway. Consistent with these findings, an increase in ERK1/2 phosphorylation was detected with Tyro3 but not with Axl overexpression. In contrast, activation of Axl stimulated the PI(3)K pathway, which was mitigated by co-expression of Tyro3. The overexpression of Tyro3 enhanced Gas6-mediated Axl phosphorylation, which was not detected upon overexpression of a "kinase dead" form of Tyro3 (kdTyro3). In addition, the overexpression of Axl induced kdTyro3 phosphorylation. Co-immunoprecipitation experiments confirmed that the Axl and Tyro3 receptors are closely associated. These findings show that overexpression of Tyro3 in the presence of Axl promotes cell proliferation, and that co-expression of Axl and Tyro3 can affect the outcome of Gas6-initiated signaling. Furthermore, they demonstrate a functional interaction between the members of the TAM receptor family which can shed light on the molecular mechanisms underlying the functional consequences of TAM receptor activation in cell transformation, neural function, immune function, and reproductive function among others.

Functional characterization of putative cilia genes by high-content analysis.

Cilia are microtubule-based protrusions from the cell surface that are involved in a number of essential signaling pathways, yet little is known about many of the proteins that regulate their structure and function. A number of putative cilia genes have been identified by proteomics and comparative sequence analyses, but functional data are lacking for the vast majority. We therefore monitored the effects in three cell lines of small interfering RNA (siRNA) knockdown of 40 of these genes by high-content analysis. We assayed cilia number, length, and transport of two different cargoes (membranous serotonin receptor 6-green fluorescent protein [HTR6-GFP] and the endogenous Hedgehog [Hh] pathway transcription factor Gli3) by immunofluorescence microscopy; and cilia function using a Gli-luciferase Hh signaling assay. Hh signaling was most sensitive to perturbations, with or without visible structural cilia defects. Validated hits include Ssa2 and mC21orf2 with ciliation defects; Ift46 with short cilia; Ptpdc1 and Iqub with elongated cilia; and Arl3, Nme7, and Ssna1 with distinct ciliary transport but not length defects. Our data confirm various ciliary roles for several ciliome proteins and show it is possible to uncouple ciliary cargo transport from cilia formation in vertebrates.

ErbB4 in parvalbumin-positive interneurons is critical for neuregulin 1 regulation of long-term potentiation.

Neuregulin 1 (NRG1) is a trophic factor that acts by stimulating ErbB receptor tyrosine kinases and has been implicated in neural development and synaptic plasticity. In this study, we investigated mechanisms of its suppression of long-term potentiation (LTP) in the hippocampus. We found that NRG1 did not alter glutamatergic transmission at SC-CA1 synapses but increased the GABA(A) receptor-mediated synaptic currents in CA1 pyramidal cells via a presynaptic mechanism. Inhibition of GABA(A) receptors blocked the suppressing effect of NRG1 on LTP and prevented ecto-ErbB4 from enhancing LTP, implicating a role of GABAergic transmission. To test this hypothesis further, we generated parvalbumin (PV)-Cre;ErbB4(-/-) mice in which ErbB4, an NRG1 receptor in the brain, is ablated specifically in PV-positive interneurons. NRG1 was no longer able to increase inhibitory postsynaptic currents and to suppress LTP in PV-Cre;ErbB4(-/-) hippocampus. Accordingly, contextual fear conditioning, a hippocampus-dependent test, was impaired in PV-Cre;ErbB4(-/-) mice. In contrast, ablation of ErbB4 in pyramidal neurons had no effect on NRG1 regulation of hippocampal LTP or contextual fear conditioning. These results demonstrate a critical role of ErbB4 in PV-positive interneurons but not in pyramidal neurons in synaptic plasticity and support a working model that NRG1 suppresses LTP by enhancing GABA release. Considering that NRG1 and ErbB4 are susceptibility genes of schizophrenia, these observations contribute to a better understanding of how abnormal NRG1/ErbB4 signaling may be involved in the pathogenesis of schizophrenia.

Kinetics of hedgehog-dependent full-length Gli3 accumulation in primary cilia and subsequent degradation.

Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. By generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitored their kinetics of accumulation in cilia upon Hh stimulation. Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilium tips is full-length and likely not protein kinase A phosphorylated. We also confirmed that phosphorylation and betaTrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than the Gli3 repressor, the latter not requiring SPOP/Cul3 or betaTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants.