4 June 2007. Genetic and biochemical evidence implicates the neuregulin 1 (NRG1) protein and its major CNS receptor, ErbB4, in schizophrenia. However, the pair is involved in so many aspects of neuronal development and adult physiology that sorting out their disease-relevant functions has been difficult.
Two papers published in the May 24 issue of Neuron reveal novel effects of the NRG1/ErbB4 signaling pathway in CNS synapses. One study shows that the pathway is essential for the proper development and function of stimulatory glutamatergic synapses. The other finds NRG1/ErbB4 is a player in the release of GABA at inhibitory synapses in the cortex.
Both of these studies have direct relevance for schizophrenia research. Glutamatergic stimulation (see Glutamate Hypothesis of Schizophrenia by Bita Moghaddam) and GABAergic inhibition (see SRF interview with David Lewis) figure largely in favored hypotheses of disease psychopathology. The new data are a step forward in connecting these genes of interest to cell and synapse function, on the way to the final goal of understanding, and ameliorating, the behavioral symptoms of the disease.
Shaping Glutamatergic Circuits
The first paper, from Bo Li and Roberto Malinow at Cold Spring Harbor Laboratory in Cold Spring Harbor, New York, in collaboration with Ran-Sook Woo and Lin Mei of Medical College of Georgia in Augusta, shows that NRG1/ErbB4 is required for the activity-dependent development and plasticity of excitatory glutamatergic synapses in the hippocampus. Their work, an elegant combination of genetic manipulations, imaging, and electrophysiology, provides an explanation for how defects in NRG1/ErbB4 signaling could lead to problems with glutamatergic circuitry.
Mei and others had previously shown that ErbB4 is located post-synaptically in excitatory synapses (Huang et al., 2000; Garcia et al., 2000), so first author Li set out to find how it got there. Using hippocampal slice cultures, Li and coworkers established that neuronal stimulation led to activation of the tyrosine kinase activity of post-synaptic ErbB4 receptors. Following activation, ErbB4 accumulated in the synapse in conjunction with PSD95. By two-photon microscopy, the researchers localized the receptor to surface spines of CA1 cells.
There, ErbB4 was no idle bystander, but played an important role in establishing synapse structure in response to activity. When the researchers overexpressed ErbB4, they found enhanced AMPA receptor currents in the neurons, and an increase in dendritic spine size. If they used RNAi to knock down ErbB4, they got suppression of AMPAR currents and lower synaptic transmission through both AMPA and NMDA receptors. RNAi also blocked synapse maturation, and reduced spine density and size. The synaptic actions of ErbB4 required an intact kinase signaling domain, and NRG1.
NRG1/ErbB4 was also involved in synaptic plasticity. Induction of LTP was accompanied by a rapid and persistent increase in ErbB4 on the surface of spines, and increased spine size. At the same time, NRG1 was detected on the surface of presynaptic boutons, and the investigators showed that levels diminished when they induced LTP, possibly because of NRG1 processing and release.
NRG1/ErbB4 modulation of synapses occurred via stabilization of AMPA receptors. If the investigators added an AMPA receptor-derived peptide that enhances retention of synaptic AMPA receptors, they no longer saw synaptic suppression with ErbB4 RNAi knockdown.
From these results, the investigators propose a model where synaptic activity leads to activation of the NRG1/ErbB4 signaling pathway, which recruits or stabilizes additional ErbB4 in the synapse. The NRG1/ErbB4 activation also stabilizes synaptic AMPA receptors, and permits synaptic plasticity and synaptic maturation. Interruption of the pathway destabilizes AMPARs and leads to impairments in plasticity and loss of spines. Decreased synapse function over time could affect development of excitatory circuits.
“Our study provides a link between NRG1 and the ‘glutamatergic hypofunction’ hypothesis as well as with the view that development of early circuitry is an important underlying factor in schizophrenia,” the authors write. The study “indicates a mechanism by which genetic deficits, developmental abnormalities and glutamatergic hypofunction can be linked together."
Pumping Up GABAergic Connections
The second paper, from Mei and colleagues at Medical College of Georgia, along with Tian-Ming Gao and coworkers at the Southern Medical University in Guangzhou, China, and several other institutions, shows a novel function for NRG1/ErbB4 in enhancing the stimulation-dependent release of GABA from inhibitory interneurons in the cortex. The results raise the possibility that changes in NRG1 activity could underlie the abnormal GABAergic neurotransmission seen in schizophrenia.
Joint first authors Ran-sook Woo and Xiao-Ming Li and colleagues show that ErbB4 messenger RNA was widely distributed in the adult rat brain, and was found in brain regions rich in interneurons. Fine structural analysis of stained sections revealed ErbB4 at presynaptic terminals of a considerable fraction of GABAergic interneurons in the prefrontal cortex.
Based on the distribution of its receptor, the researchers began to look for effects of NRG1 on GABA release in cortical slices. Treatment of slices with NRG1 caused no change in basal release, but increased release in response to depolarization. Consistent with this result, electrophysiological recording revealed that NRG1 addition did affect spontaneous miniature inhibitor post-synaptic currents (IPSCs), but enhanced evoked IPSCs.
NRG1 seemed to act directly on GABA release, as a panel of inhibitors of other neurotransmitter actions had no effect on GABA release. In support of this idea, NRG increased GABA release from isolated synaptosomes, the researchers found. Also, NRG1 lowered the amplitude of later release when cells were stimulated consecutively in a paired pulse protocol. This result suggests that NRG1 was acting to increase the fractional release of neurotransmitter.
The effects of NRG1 could be blocked by a truncated ErbB4 receptor, which acted like a sponge for NRG1, by binding ligand but performing no signaling function. Interestingly, when only the ecto-ErbB4 was added to the slice cultures, it inhibited evoked GABA release. This suggests that endogenous NRG1 plays a role in GABA release.
Other data pointed to ErbB4 as the relevant receptor in the cortical slices. The NRG1-induced increase in synaptic activity was inhibited by evoked ErbB4 kinase inhibitors. Finally, in cells from ErbB4 mutant mice, NRG1 had no effect.
The results identify a novel function of NRG1 in regulating GABAergic transmission via presynaptic ErbB4 receptors. “These results suggest that NRG1 may regulate the activity of cortical interneurons, providing insight into potential mechanisms by which this trophic factor regulates synaptic plasticity and pathogeneses of schizophrenia and epilepsy,” the authors write.
Brain out of Balance
In an accompanying commentary, Gerald Fischbach of Columbia University in New York offers a model for how these results might fit into schizophrenia. “In one speculative scenario, the schizophrenia risk haplotypes might result in NRG1 hypofunction, leading to a decrease in the efficacy of glutamate and GABA-mediated synaptic transmission in the prefrontal cortex, which could produce desynchronized firing of pyramidal neurons, the loss of gamma waves recorded on the brain surface, and behavioral deficits in working memory, an important hallmark of schizophrenia.”
He continues, “Such oversimplified schemes have their uses, in that they allow one to think about disorganized thoughts in an organized way, and illustrate the paths one might hope to follow in a bottom-up approach to understanding complex mental and behavioral phenomena.”
The work also highlights many unanswered questions. One outstanding unknown is how schizophrenia-associated polymorphisms in NRG1 and ErbB4 genes affect that pathway's function (see SRF related news story). In addition, only a fraction of people with schizophrenia have NRG1 or ErbB4 mutations, so the question remains of how the pathway might be affected in the others.
Fischbach concludes, “This work takes us one step closer to understanding the synaptic transmission and local circuit deficits in schizophrenia, but exactly how and to what extent these defects may contribute to the characteristic disease symptoms and etiology is a challenge for future research.”—Pat McCaffrey.
Li B, Woo RS, Mei L, Malinow R. The Neuregulin-1 Receptor ErbB4 Controls Glutamatergic Synapse Maturation and Plasticity. Neuron. 2007 May 24;54(4):583-97. Abstract
Woo RS, Li XM, Tao Y, Carpenter-Hyland E, Huang YZ, Weber J, Neiswender H, Dong XP, Wu J, Gassmann M, Lai C, Xiong WC, Gao TM, Mei L. Neuregulin-1 Enhances Depolarization-Induced GABA Release. Neuron. 2007 May 24;54(4):599-610. Abstract
Fischbach GD. NRG1 and Synaptic Function in the CNS. Neuron. 2007 May 24;54(4):495-7. Abstract