Yisheng Lu

Neuregulin 1 regulates pyramidal neuron activity via ErbB4 in parvalbumin-positive interneurons

Neuregulin 1 (NRG1) is a trophic factor thought to play a role in neural development. Recent studies suggest that it may regulate neurotransmission, mechanisms of which remain elusive. Here we show that NRG1, via stimulating GABA release from interneurons, inhibits pyramidal neurons in the prefrontal cortex (PFC). Ablation of the NRG1 receptor ErbB4 in parvalbumin (PV)-positive interneurons prevented NRG1 from stimulating GABA release and from inhibiting pyramidal neurons. PV-ErbB4−/− mice exhibited schizophrenia-relevant phenotypes similar to those observed in NRG1 or ErbB4 null mutant mice, including hyperactivity, impaired working memory, and deficit in prepulse inhibition (PPI) that was ameliorated by diazepam, a GABA enhancer. These results indicate that NRG1 regulates the activity of pyramidal neurons by promoting GABA release from PV-positive interneurons, identifying a critical function of NRG1 in balancing brain activity. Because both NRG1 and ErbB4 are susceptibility genes of schizophrenia, our study provides insight into potential pathogenic mechanisms of schizophrenia and suggests that PV-ErbB4−/− mice may serve as a model in the study of this and relevant brain disorders.

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 GABAA receptor-mediated synaptic currents in CA1 pyramidal cells via a presynaptic mechanism. Inhibition of GABAA 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.

Distinct Roles of Muscle and Motoneuron LRP4 in Neuromuscular Junction Formation

Neuromuscular junction (NMJ) formation requires precise interaction between motoneurons and muscle fibers. LRP4 is a receptor of agrin that is thought to act in cis to stimulate MuSK in muscle fibers for postsynaptic differentiation. Here we dissected the roles of LRP4 in muscle fibers and motoneurons in NMJ formation by cell-specific mutation. Studies of muscle-specific mutants suggest that LRP4 is involved in deciding where to form AChR clusters in muscle fibers, postsynaptic differentiation, and axon terminal development. LRP4 in HEK293 cells increased synapsin or SV2 puncta in contacting axons of cocultured neurons, suggesting a synaptogenic function. Analysis of LRP4 muscle and motoneuron double mutants and mechanistic studies suggest that NMJ formation may also be regulated by LRP4 in motoneurons, which could serve as agrins receptor in trans to induce AChR clusters. These observations uncovered distinct roles of LRP4 in motoneurons and muscles in NMJ development.

Reversal of behavioral deficits and synaptic dysfunction in mice overexpressing neuregulin 1.

Neuregulin 1 (Nrg1) is a susceptibility gene of schizophrenia, a disabling mental illness that affects 1% of the general population. Here, we show that ctoNrg1 mice, which mimic high levels of NRG1 observed in forebrain regions of schizophrenic patients, exhibit behavioral deficits and hypofunction of glutamatergic and GABAergic pathways. Intriguingly, these deficits were diminished when NRG1 expression returned to normal in adult mice, suggesting that damage which occurred during development is recoverable. Conversely, increase of NRG1 in adulthood was sufficient to cause glutamatergic impairment and behavioral deficits. We found that the glutamatergic impairment by NRG1 overexpression required LIM domain kinase 1 (LIMK1), which was activated in mutant mice, identifying a pathological mechanism. These observations demonstrate that synaptic dysfunction and behavioral deficits in ctoNrg1 mice require continuous NRG1 abnormality in adulthood, suggesting that relevant schizophrenia may benefit from therapeutic intervention to restore NRG1 signaling.Neuregulin 1 (Nrg1) is a susceptibility gene of schizophrenia, a disabling mental illness that affects 1% of the general population. Here, we show that ctoNrg1 mice, which mimic high levels of NRG1 observed in forebrain regions of schizophrenic patients, exhibit behavioral deficits and hypofunction of glutamatergic and GABAergic pathways. Intriguingly, these deficits were diminished when NRG1 expression returned to normal in adult mice, suggesting that damage which occurred during development is recoverable. Conversely, increase of NRG1 in adulthood was sufficient to cause glutamatergic impairment and behavioral deficits. We found that the glutamatergic impairment by NRG1 overexpression required LIM domain kinase 1 (LIMK1), which was activated in mutant mice, identifying a pathological mechanism. These observations demonstrate that synaptic dysfunction and behavioral deficits in ctoNrg1 mice require continuous NRG1 abnormality in adulthood, suggesting that relevant schizophrenia may benefit from therapeutic intervention to restore NRG1 signaling.

Antibodies against low-density lipoprotein receptor–related protein 4 induce myasthenia gravis

Myasthenia gravis (MG) is the most common disorder affecting the neuromuscular junction (NMJ). MG is frequently caused by autoantibodies against acetylcholine receptor (AChR) and a kinase critical for NMJ formation, MuSK; however, a proportion of MG patients are double-negative for anti-AChR and anti-MuSK antibodies. Recent studies in these subjects have identified autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), an agrin receptor also critical for NMJ formation. LRP4 autoantibodies have not previously been implicated in MG pathogenesis. Here we demonstrate that mice immunized with the extracellular domain of LRP4 generated anti-LRP4 antibodies and exhibited MG-associated symptoms, including muscle weakness, reduced compound muscle action potentials (CMAPs), and compromised neuromuscular transmission. Additionally, fragmented and distorted NMJs were evident at both the light microscopic and electron microscopic levels. We found that anti-LRP4 sera decreased cell surface LRP4 levels, inhibited agrin-induced MuSK activation and AChR clustering, and activated complements, revealing potential pathophysiological mechanisms. To further confirm the pathogenicity of LRP4 antibodies, we transferred IgGs purified from LRP4-immunized rabbits into naive mice and found that they exhibited MG-like symptoms, including reduced CMAP and impaired neuromuscular transmission. Together, these data demonstrate that LRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood.

LRP4 Is Critical for Neuromuscular Junction Maintenance

The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms.

Maintenance of GABAergic Activity by Neuregulin 1-ErbB4 in Amygdala for Fear Memory

Inhibitory neurotransmission in amygdala is important for fear learning and memory. However, mechanisms that control the inhibitory activity in amygdala are not well understood. We provide evidence that neuregulin 1 (NRG1) and its receptor ErbB4 tyrosine kinase are critical for maintaining GABAergic activity in amygdala. Neutralizing endogenous NRG1, inhibition, or genetic ablation of ErbB4, which was expressed in a majority of palvalbumin (PV)+ neurons in amygdala, reduced GABAergic transmission and inhibited tone-cued fear conditioning. Specific ablation of ErbB4 in PV+ neurons reduced eIPSC/eEPSC ratios and impaired fear conditioning. Notably, expression of ErbB4 in amygdala was sufficient to diminish synaptic dysfunction and fear conditioning deficits in PV-ErbB4-/- mice. These observations indicated that NRG1 signaling maintains high GABAergic activity in amygdala and, thus, regulates fear memory. Considering that both NRG1 and ErbB4 are susceptibility genes of schizophrenia, our study sheds light on potential pathophysiological mechanisms of this disorder.

The AKAP Yu is required for olfactory long-term memory formation in Drosophila

Extensive neurogenetic analysis has shown that memory formation depends critically on cAMP-protein kinase A (PKA) signaling. Details of how this pathway is involved in memory formation, however, remain to be fully elucidated. From a large-scale behavioral screen in Drosophila, we identified the yu mutant to be defective in one-day memory after spaced training. The yu mutation disrupts a gene encoding an A-kinase anchoring protein (AKAP). AKAPs comprise a family of proteins, which determine the subcellular localization of PKAs and thereby critically restrict cAMP signaling within a cell. Further behavioral characterizations revealed that long-term memory (LTM) was disrupted specifically in the yu mutant, whereas learning, short-term memory and anesthesia-resistant memory all appeared normal. Another independently isolated mutation of the yu gene failed to complement the LTM defect associated with the yu mutation, and this phenotypic defect could be rescued by induced acute expression of a yu+ transgene, suggesting that yu functions physiologically during memory formation. AKAP Yu is expressed preferentially in the mushroom body (MB) neuroanatomical structure, and expression of a yu+ transgene to the MB, but not to other brain regions, is sufficient to rescue the LTM defect of the yu mutant. These observations lead us to conclude that proper localization of PKA by Yu AKAP in MB neurons is required for the formation of LTM.

Slit2 as a β-catenin/Ctnnb1-dependent retrograde signal for presynaptic differentiation

Neuromuscular junction formation requires proper interaction between motoneurons and muscle cells. β-Catenin (Ctnnb1) in muscle is critical for motoneuron differentiation; however, little is known about the relevant retrograde signal. In this paper, we dissected which functions of muscle Ctnnb1 are critical by an in vivo transgenic approach. We show that Ctnnb1 mutant without the transactivation domain was unable to rescue presynaptic deficits of Ctnnb1 mutation, indicating the involvement of transcription regulation. On the other hand, the cell-adhesion function of Ctnnb1 is dispensable. We screened for proteins that may serve as a Ctnnb1-directed retrograde factor and identified Slit2. Transgenic expression of Slit2 specifically in the muscle was able to diminish presynaptic deficits by Ctnnb1 mutation in mice. Slit2 immobilized on beads was able to induce synaptophysin puncta in axons of spinal cord explants. Together, these observations suggest that Slit2 serves as a factor utilized by muscle Ctnnb1 to direct presynaptic differentiation. DOI: http://dx.doi.org/10.7554/eLife.07266.001

Amygdala NRG1-ErbB4 is critical for the modulation of anxiety-like behaviors.

Anxiety disorder is related to the pathophysiology of psychiatric diseases, including major depression, substance abuse, and schizophrenia. The amygdala is important for manifestation and modulation of anxiety. However, relatively little is known regarding the mechanisms that control the amygdala inhibitory activity that is involved in anxiety. We found that almost all ErbB4, which is the only autonomous receptor of neuregulin 1 (NRG1) in the basolateral amygdala (BLA), was expressed in GABAergic neurons. Endogenous NRG1–ErbB4 signaling pathway in the BLA could modulate anxiety-like behaviors and GABA release, whereas it had no effect on glutamatergic transmission. The administration of NRG1 into the BLA of high-anxiety mice alleviated their anxiety and enhanced GABAergic neurotransmission. Moreover, exogenous NRG1 also produced an anxiolytic effect in the stressed mice. Together, these observations indicated that NRG1–ErbB4 signaling is critical to maintaining GABAergic activity in the amygdala and thus to modulating anxiety-like behaviors. Because NRG1 and ErbB4 are susceptibility genes of schizophrenia, our findings might also help to explain the potential mechanism of emotional abnormality in schizophrenia.