6 March 2009. A new study counters prevailing beliefs about the role of neuregulin-1 (NRG1) in the central nervous system. Some researchers deem the gene that encodes it key to understanding how schizophrenia develops, but mice that lack the genes for it or its ErbB2 or ErbB4 receptors cannot live, hampering efforts to study it in vivo. However, when Ulrich Müller of the Scripps Research Institute in La Jolla, California, and his colleagues canceled NRG1 signaling by engineering mice that lacked ErbB2 and ErbB4 receptors specifically in the central nervous system, the mice survived with brains that, at first glance, appeared normal. A closer look revealed changes, at different phenotypic levels, resembling those seen in schizophrenia, including poorly developed dendritic spines, aggressive social behavior, and impaired sensory gating. As reported in PNAS online on February 24, clozapine, an atypical antipsychotic, normalized the dendrites and behavior.
NRG1 enables the nervous system to develop and function normally (see
Taveggia et al., 2005). Its apparent jobs include sending different cell types on their separate developmental paths and helping synapses to function (see SRF related news story; SRF news story; SRF news story). In addition, it may regulate neurotransmitter receptors, including the N-methyl-D-aspartate (NMDA) receptors that play a prominent role in the glutamate hypothesis of schizophrenia (see SRF current hypotheses by B. Moghaddam; D. Javitt). NRG1 launches tyrosine kinase signaling by binding to its receptors, which consists of dimers containing ErbB2 and ErbB4, the only ErbBs essential to this pathway, although ErbB3 may play a supporting role.
Müller, first author Claudia Barros, also of the Scripps Research Institute in La Jolla, and their colleagues crossed mice that were homozygous for loxP-flanked (flox) ErbB2 and ErbB4 alleles with hGFAP-CRE mice to create what they dubbed ErbB2/B4-CNSko mice. They compared them with littermates that lacked the CRE or floxed alleles; as far as they could tell, these control mice did not differ from wild-type mice.
Despite the absence of ErbB2/ErbB4 proteins, the brains of the ErbB2/B4-CNSko mice grew to the usual size, with normal layers in the cerebral cortex, hippocampus, and cerebellum. “These findings were unexpected, as NRG1/ErbB signaling was thought to be essential for the formation of cortical cell layers,” write Barros and associates.
Up to that point, the investigators had simply been trying to confirm the role of NRG1/ErbB in the migration of neurons into the cerebral cortex and to understand the mechanisms involved. When they stumbled upon these findings, they realized that their implications for schizophrenia begged for further study.
Serendipity leads to postnatal functions of NRG1
Barros and colleagues followed the data trail. They checked to see if the lack of NRG1/ErbB signaling affected the development of dendritic spines (see SRF related news story), finger-like projections on the ends of nerve cells that relay messages between neurons, in the cortex and hippocampus. Although dendrites in the hippocampus and cortex of the ErbB2/4 knockout mice had formed into a fine shape overall, dendritic spines were sparsely scattered compared to those of control mice. These abnormalities resemble those seen in humans with schizophrenia (Law et al., 2004).
To learn how the differences between the two groups of mice arose, Barros and colleagues cultured hippocampal neurons and waited for dendritic spines to grow. After 11 days, cells from the ErbB knockout mice had fallen behind those of controls. By day 21, they not only had half the usual number of dendritic spines on mature neurons, those they did have were thinner. Furthermore, staining to detect glutamate transporter at the synapses revealed fewer excitatory presynaptic nerve endings in the ErbB2/4 knockout mice compared to the control group.
Barros and associates were able to rule out cell death as the cause of the spine abnormalities; instead, they found further evidence tying them to the absent NRG1 signaling. When they added recombinant NRG1 to wild-type hippocampal neurons in culture, dendritic spines and excitatory presynaptic nerve endings proliferated. According to the researchers, this suggests that NRG1 signaling, through ErbB2/ErbB4, controls the formation of mature dendritic spines and excitatory presynaptic nerve terminals in the mouse hippocampus.
Delving more deeply into the mechanisms, the researchers suspected the involvement of postsynaptic density 95 (PSD-95), a protein that binds to and colocalizes with NMDA receptors (see SRF related news story). Sure enough, they found that NMDA receptors and PSD-95 congregated together less in the ErbB2/4 knockout mice, despite normal levels of both. They think that interactions of PSD-95 with ErbB4 and, indirectly, ErbB2, followed by binding of PSD-95 with NMDA receptors, enable dendritic spines to incorporate glutamate receptors, a healthy developmental step.
Important for schizophrenia?
The differences between the ErbB2/4 knockout and control mice did not stop at the brain; the two groups behaved differently in tests that elicit behaviors relevant to schizophrenia. For instance, in the resident-intruder paradigm, the ErbB knockouts acted more persistently aggressive toward other mice. Furthermore, male, but not female, mutant mice, showed decreased sensory gating in the prepulse inhibition test, echoing findings from studies of people with schizophrenia. Administering clozapine (see SRF related news story) reversed these behavioral differences and returned the dendritic spines of the Erb2/4 knockout mice to normal.
Barros and colleagues write, “Our findings challenge the current view of the role of NRG1/ErbB signaling in the CNS.” Specifically, it does not seem critical for the normal development of cell layers in the brain. In an interview with SRF, Müller says the prevailing wisdom about the functions of NRG1 signaling in the central nervous system comes from studies done in vitro or in mice with incompletely cancelled NRG1 signaling; results of the former may not apply to the whole organism, and the latter cannot rule out other signaling paths. He said that the new study sidestepped this problem by eliminating NRG1 signaling altogether and offers a new animal model that could facilitate the study of schizophrenia.
The researchers conclude that abnormal NRG1/ErbB signaling may foster schizophrenia by changing excitatory synapses in the brain. Their results strengthen the case for glutamate-related dysfunction in the disease.—Victoria L. Wilcox.
Barros CS, Calabrese B, Chamero P, Roberts AJ, Korzus E, Lloyd K, Stowers L, Mayford M, Halpain S, Müller U. Impaired maturation of dendritic spines without disorganization of cortical cell layers in mice lacking NRG1/ErbB signaling in the central nervous system. PNAS Early Edition. 2009 Feb 24. Abstract