Wen Cheng Xiong

Neuregulin 1 in neural development, synaptic plasticity and schizophrenia

Schizophrenia is a highly debilitating mental disorder that affects ∼1% of the general population, yet it continues to be poorly understood. Recent studies have identified variations in several genes that are associated with this disorder in diverse populations, including those that encode neuregulin 1 (NRG1) and its receptor ErbB4. The past few years have witnessed exciting progress in our knowledge of NRG1 and ErbB4 functions and the biological basis of the increased risk for schizophrenia that is potentially conferred by polymorphisms in the two genes. An improved understanding of the mechanisms by which altered function of NRG1 and ErbB4 contributes to schizophrenia might eventually lead to the development of more effective therapeutics.

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.

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.

Retrograde regulation of motoneuron differentiation by muscle β-catenin

Synapse formation requires proper interaction between pre- and postsynaptic cells. In anterograde signaling, neurons release factors to guide postsynaptic differentiation. However, less is known about how postsynaptic targets retrogradely regulate presynaptic differentiation or function. We found that muscle-specific conditional knockout of β-catenin (Ctnnb1, also known as β-cat) in mice caused both morphologic and functional defects in motoneuron terminals of neuromuscular junctions (NMJs). In the absence of muscle β-catenin, acetylcholine receptor clusters were increased in size and distributed throughout a wider region. Primary nerve branches were mislocated, whereas secondary or intramuscular nerve branches were elongated and reduced in number. Both spontaneous and evoked neurotransmitter release was reduced at the mutant NMJs. Furthermore, short-term plasticity and calcium sensitivity of neurotransmitter release were compromised in β-catenin–deficient muscle. In contrast, the NMJ was normal in morphology and function in motoneuron-specific β-catenin–deficient mice. Taken together, these observations indicate a role for muscle β-catenin in presynaptic differentiation and function, identifying a previously unknown retrograde signaling in the synapse formation and synaptic plasticity.

β-Catenin Regulates Acetylcholine Receptor Clustering in Muscle Cells through Interaction with Rapsyn

Agrin is believed to be a factor used by motoneurons to direct acetylcholine receptor (AChR) clustering at the neuromuscular junction. However, exactly how agrin mediates this effect remains unclear. Here we demonstrate that the β-catenin interacts with rapsyn, a molecule key for AChR clustering. Agrin stimulation increases the association of β-catenin with surface AChRs. Suppression of β-catenin expression inhibited agrin-induced AChR clustering, suggesting a necessary role of β-catenin in this event. The β-catenin action did not appear to require the function of T-cell factors (TCFs), suggesting a mechanism independent of TCF-mediated transcription. In contrast, prevention of β-catenin from interacting with α-catenin attenuated agrin-induced AChR clustering. These results suggest that β-catenin may serve as a link between AChRs and α-catenin-associated cytoskeleton, revealing a novel function of β-catenin in synaptogenesis.

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 erbinΔC/ΔC 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.

Neuromuscular Junction Formation, Aging, and Disorders

Synapses, the fundamental unit in neuronal circuits, are critical for learning and memory, perception, thinking, and reaction. The neuromuscular junction (NMJ) is a synapse formed between motoneurons and skeletal muscle fibers that is covered by Schwann cells (SCs). It is essential for controlling muscle contraction. NMJ formation requires intimate interactions among motoneurons, muscles, and SCs. Deficits in NMJ formation and maintenance cause neuromuscular disorders, including congenital myasthenic syndrome and myasthenia gravis. NMJ decline occurs in aged animals and may appear before clinical presentation of motoneuron disorders such as amyotrophic lateral sclerosis. We review recent findings in NMJ formation, maintenance, neuromuscular disorders, and aging of the NMJ, focusing on communications among motoneurons, muscles and SCs, and underlying mechanisms.

FAK interaction with MBD2: A link from cell adhesion to nuclear chromatin remodeling?

Cell adhesion, migration, proliferation, and differentiation are tightly linked and coordinated cellular processes. Cell adhesion dependent gene expression is believed to contribute to such coordination. Focal adhesion kinase (FAK) and its related protein, PYK2 (proline rich tyrosine kinase 2), are a major family of cell adhesion activated tyrosine kinases that play important roles in these cellular processes. Whereas FAK or PYK2 is known to be a scaffold protein, recruiting many cytoplasmic proteins into the focal adhesion complex and regulating focal adhesion turnover and cell migration, how FAK or PYK2 links to the nuclei and regulates gene expression remain largely unclear. We recently report a new signaling of FAK in regulating heterochromatin remodeling by its interaction with MBD2 (Methyl CpG binding domain protein 2), which may underlie FAK regulation of myogenin expression and muscle differentiation. Two insights have been obtained through the analysis of FAK-MBD2 interaction. The interaction appears to be sufficient, but not necessary, for FAK translocation into or maintaining in the nucleus. The nuclear FAK-MBD2 complexes cause altered heterochromatin organization and decreased MBD2 association with HDAC1 (histone deacetylase complex 1) and methyl CpG site in the myogenin promoter, thus, inducing myogenin expression. These results demonstrate a new mechanism underlying FAK regulation of gene expression, and suggest a potential link between cell adhesion and cell differentiation.v

Calcyon stimulates neuregulin 1 maturation and signaling

Neuregulin1 (NRG1) is a single transmembrane protein that plays a critical role in neural development and synaptic plasticity. Both NRG1 and its receptor, ErbB4, are well-established risk genes of schizophrenia. The NRG1 ecto-domain (ED) binds and activates ErbB4 following proteolytic cleavage of pro-NRG1 precursor protein. Although several studies have addressed the function of NRG1 in brain, very little is known about the cleavage and shedding mechanism. Here we show that the neuronal vesicular protein calcyon is a potent activator and key determinant of NRG1 ED cleavage and shedding. Calcyon stimulates clathrin-mediated endocytosis and endosomal targeting; and its levels are elevated in postmortem brains of schizophrenics. Overexpression of calcyon stimulates NRG1 cleavage and signaling in vivo, and as a result, GABA transmission is enhanced in calcyon overexpressing mice. Conversely, NRG1 cleavage, ErbB4 activity and GABA transmission are decreased in calcyon null mice. Moreover, stimulation of NRG1 cleavage by calcyon was recapitulated in HEK 293 cells suggesting the mechanism involved is cell-autonomous. Finally, studies with site-specific mutants in calcyon and inhibitors for the major sheddases indicate that the stimulatory effects of calcyon on NRG1 cleavage and shedding depend on clathrin-mediated endocytosis, β-secretase 1, and interaction with clathrin adaptor proteins. Together these results identify a novel mechanism for NRG1 cleavage and shedding.

Increased microglial activity, impaired adult hippocampal neurogenesis, and depressive-like behavior in microglial VPS35 depleted mice

Vacuolar sorting protein 35 (VPS35) is a critical component of retromer, which is essential for selective endosome-to-Golgi retrieval of membrane proteins. VPS35 deficiency is implicated in neurodegenerative disease pathology, including Alzheimers disease (AD). However, exactly how VPS35 loss promotes AD pathogenesis remains largely unclear. VPS35 is expressed in various types of cells in the brain, including neurons and microglia. Whereas neuronal VPS35 plays a critical role in preventing neurodegeneration, the role of microglial VPS35 is largely unknown. Here we provide evidence for microglial VPS35s function in preventing microglial activation and promoting adult hippocampal neurogenesis. VPS35 is expressed in microglia in various regions of the mouse brain, with a unique distribution pattern in a brain region-dependent manner. Conditional knocking out of VPS35 in microglia of male mice results in regionally increased microglial density and activity in the subgranular zone of the hippocampal dentate gyrus (DG), accompanied by elevated neural progenitor proliferation, but decreased neuronal differentiation. Additionally, newborn neurons in the mutant DG show impaired dendritic morphology and reduced dendritic spine density. When examining the behavioral phenotypes of these animals, microglial VPS3S-depleted mice display depression-like behavior and impairment in long-term recognition memory. At the cellular level, VPS35-depleted microglia have grossly enlarged vacuolar structures with increased phagocytic activity toward postsynaptic marker PSD95, which may underlie the loss of dendritic spines observed in the mutant DG. Together, these findings identify an important role of microglial VPS35 in suppressing microglial activation and promoting hippocampal neurogenesis, which are both processes involved in AD pathogenesis. SIGNIFICANCE STATEMENT The findings presented here provide the first in vivo evidence that Vacuolar sorting protein 35 (VPS35)/retromer is essential for regulating microglial function and that when microglial retromer mechanics are disrupted, the surrounding brain tissue can be affected in a neurodegenerative manner. These findings present a novel, microglial-specific role of VPS35 and raise multiple questions regarding the mechanisms underlying our observations. These findings also have myriad implications for the field of retromer research and the role of retromer dysfunction in neurodegenerative pathophysiology. Furthermore, they implicate a pivotal role of microglia in the regulation of adult hippocampal neurogenesis and the survival/integration of newborn neurons in the adult hippocampus.