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Interneurons Are Stage for Neuregulin 1, ErbB4 Interplay

14 April 2010. A study published online in Nature today, in combination with a paper from earlier this year in PNAS, points to parvalbumin-containing interneurons as a key venue for neuregulin-1 (NRG1) and ErbB4 signaling in the cerebral cortex. These papers put attention squarely on the brain's inhibitory circuits when considering the function of NRG1 and its receptor ErbB4, which are encoded by genes that are candidate susceptibility factors for schizophrenia (see SZGene for NRG1 and ErbB4).

From the labs of Oscar Marín and Beatriz Rico at Universidad Miguel Hernández in Sant Joan d’Alacant, Spain, the study in Nature finds that NRG1-ErbB4 signaling controls the wiring diagrams made by interneurons during brain development. The researchers localized the ErbB4 receptor to parvalbumin-containing interneurons in several regions of the brain. They further pinpointed ErbB4 to both the axon terminals and the dendrites of interneurons, and found that the receptor promotes synapse formation on both ends. Similar to other results (Vullhorst et al., 2009), the researchers did not turn up much evidence for NRG1-ErbB4 signaling in excitatory pyramidal neurons.

Beyond development, NRG1-ErbB4 signaling could modulate fully formed synapses, according to the January 19, 2010, PNAS paper from Lin Mei and colleagues at Medical College of Georgia in Augusta. The researchers found that application of NRG1 protein slowed action potential firing of excitatory pyramidal cells, and this dampening was likely mediated by ErbB4 receptors and the inhibitory neurotransmitter GABA found inside parvalbumin-containing interneurons. The researchers also found behavioral anomalies reminiscent of schizophrenia in mice lacking ErbB4 in parvalbumin-containing neurons.

Chandeliers and baskets
Parvalbumin, a calcium binding protein, marks a subset of interneurons that are fast-spiking cells, capable of sustaining rapid-fire action potentials. These types of interneurons have a hand in creating gamma waves, a kind of synchronized brain activity reported to be disrupted in people with schizophrenia (Spencer et al., 2003; see SRF hypothesis paper by Woo et al.). This connection to schizophrenia dovetails with an older line of research stemming from the postmortem observation of protein expression alteration in some populations of interneurons in cortex of people with schizophrenia (see SRF interview with David Lewis).

In their Nature paper, first author Pietro Fazzari and colleagues took a multi-pronged approach to pinpoint the location of NRG1-ErbB4 signaling to interneurons. Using mice engineered to have all their interneurons labeled with GFP, they first found that most ErbB4-expressing cells were, in fact, interneurons, and this pattern held for multiple cortical regions, including motor cortex, somatosensory cortex, visual cortex, and hippocampus. Next, they found that ErbB4 resided mainly in parvalbumin-containing neurons: for example, 85 percent of parvalbumin cells in the prefrontal cortex were also positive for ErbB4 expression, whereas only 10 percent of cells containing calretinin, a marker for a different class of interneuron, were positive for ErbB4. These ErbB4-positive neurons usually took on the distinctive shapes of chandelier and basket cells, which are fast-spiking interneurons. Finally, electron microscopy localized gold-tagged ErbB4 to the presynaptic boutons of interneurons contacting excitatory pyramidal cells, and to the post-synaptic densities along the dendrites of interneurons receiving excitatory input.

Fazzari and colleagues then found that NRG1-ErbB4 signaling promoted inhibitory synapse formation. Using electroporation to introduce genetic constructs to perturb NRG1-ErbB4 signaling during embryonic brain development, the researchers report that overexpressing NRG1 led to nearly twice as many GABA-containing boutons, whereas ablating ErbB4 decreased the density of boutons made by chandelier cells.

The researchers found that eliminating Erb4 specifically from interneurons in mice also translated into functional changes: pyramidal cells in the hippocampus of these mice received fewer spontaneous deliveries of GABA ("minis," in electrophysiologist lingo) than did those receiving input from interneurons containing ErbB4 from control mice. This suggests that an interneuron lacking ErbB4 has a pre-synaptic bouton that is not operating at full capacity. As for the post-synaptic side, interneurons lacking ErbB4 also received fewer minis from excitatory inputs than normal, and had a decreased density of glutamatergic terminals contacting them. Together, these results suggest that ErbB4 influences the intricacies of neural wiring during development via its role in the formation or maintenance of inhibitory synapses onto excitatory cells, or of excitatory synapses onto inhibitory cells.

A damper on excitement
Looking beyond development, the PNAS paper explored the function of NRG1-ErbB4 signaling on fully formed synapses. Prompted by their earlier work finding that NRG1 enhances GABA release in the cortex (Woo et al., 2007), Lin Mei's team examined the acute effects of NRG1 on excitatory pyramidal cells, which are contacted by interneurons. Sure enough, first author Lei Wen and colleagues found that adding NRG1 to brain slices made from the prefrontal cortex dampened activity—within five minutes—recorded from the excitatory pyramidal cells. The effect was dose dependent, with the highest dose of NRG1 reducing firing rates to 75 percent of normal levels. Blocking ErbB4 receptors or GABAA receptors prevented this effect, indicating that these receptors mediated NRG1's dampening action.

Because previous studies have placed ErbB4 inside parvalbumin-containing neurons (e.g., Fisahn et al., 2009), Wen and colleagues hypothesized that this type of interneuron was behind the NRG1-mediated decrease in pyramidal cell firing. To test this, they engineered mice in which ErbB4 was selectively eliminated from parvalbumin-containing neurons. NRG1 was no longer able to inhibit pyramidal neuron firing in brain slices made from these mice; similarly, NRG1 could no longer enhance the inhibitory synaptic currents received by pyramidal cells. These results suggest that NRG1 normally activates ErbB4 receptors in parvalbumin-containing interneurons, which then release more GABA onto pyramidal cells, reducing their activity.

The researchers also examined the behavior of mice lacking ErbB4 in parvalbumin-containing interneurons, and though they appeared normal in several respects, some schizophrenia-like features were detected. The mice were hyperactive, demonstrated poorer working memory in a radial arm maze task, and exhibited impaired paired-pulse inhibition—a measure of how much a startle response to a loud sound is diminished by a preceding tone. This deficit could be remedied with diazepam, a drug that enhances signals through GABA receptors, which is consistent with reduced inhibitory signaling in these mice.

Though both studies put interneurons at the center of NRG1-ErbB4 signaling in the brain both during development and afterwards, perturbations to inhibitory circuitry may well radiate out to disrupt other neurotransmitter systems. And because different brain regions probably consist of slightly different circuitries, future studies will have to settle the extent to which these new results hold in different brain areas. Overall, these studies continue the hard—yet crucial—work of examining how specific brain circuits are influenced by schizophrenia risk factors.—Michele Solis.

References:
Fazzari P, Paternain AV, Valiente M, Pla R, Luján R, Lloyd K, Lerma J, Marín O, Rico B. Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling. Nature 2010 April 15.

Wen L, Lu YS, Zhu XH, Li XM, Woo RS, Chen YJ, Yin DM, Lai C, Terry AV, Vazdarjanova A, Xiong WC, Mei L. Neuregulin 1 regulates pyramidal neuron activity via ErbB4 in parvalbumin-positive interneurons. Proc Natl Acad Sci USA. 2010 Jan; 107: 1211-1216. Abstract

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Related News: Unkind Cuts of NRG3 May Lead to Schizophrenia

Comment by:  Assen Jablensky
Submitted 15 September 2010
Posted 15 September 2010

Common or rare genetic variation in NRG3 influences risk for schizophrenia?
Emerging evidence implicating NRG3 as a likely susceptibility gene in population samples as diverse as the Ashkenazi Jews, Han Chinese, Australians of Anglo-Irish ancestry, and white Americans is certainly a “noteworthy” occurrence in schizophrenia genetics. The latest addition to the evidence (Kao et al., 2010) provides considerable support to earlier (Fallin et al., 2003; Wang et al., 2008) and recent findings of association of several polymorphisms (rs10883866, rs6584400, rs10748842) within a conserved linkage disequilibrium (LD) block in intron 1 of the NRG3 gene with a delusion-laden factor and a neurocognitive quantitative trait in the schizophrenia phenotype (Chen et al., 2009; Morar et al., 2010).

A fundamental contribution of the present study is the cloning and detailed characterization of full-length NRG3 transcripts from postmortem fetal, child, adolescent, and adult brain samples (whole brain, hippocampus, and dorsolateral prefrontal cortex). Sequencing of the cDNA clones and expression analysis revealed a complex picture of alternative splicing, abundance of developmentally regulated transcripts in schizophrenia brains, and, notably, increased expression of a fetal brain-derived clone (hFBNRG3), which introduces a premature stop codon resulting in a truncated protein and a possibly destabilized NRG3-ErbB4 signalling pathway. In their clinical collections (a family-based sample and a partially independent case-control sample), the authors report significant associations of rs10748842 (representing 12 SNPs located in the LD block within intron 1) with schizophrenia, with the PANSS (Positive and Negative Syndrome Scale) subscale score on delusion severity, as well as with the PANSS negative symptom load.

Overall, the findings from this investigation and the earlier studies appear to be in a broad agreement, converging on a plausible role of NRG3 in schizophrenia pathogenesis. However, there is a fly in the ointment: The associations found in the present study exhibit a risk allele reversal compared to previously reported results; namely, all significant associations are with the major, common alleles, rather than with the minor alleles, as in Chen et al. (2009) and Morar et al. (2010). While many reasons for genuine allele flipping can be invoked (multi-locus interactions, variation in local patterns of LD, environmental exposures, ethnic background differences—see Clarke and Cardon, 2010), the explanation for the flip in this particular context is not obvious, and NRG3 should remain on the examination bench. Even in the GWAS era, studies proceeding from biologically and clinically anchored hypotheses remain rewarding and potentially productive.

References:

Chen PL, Avramopoulos D, Lasseter VK, McGrath JA, Fallin MD, Liang K-Y, Nestadt G, Feng N, Steel G, Cutting AS, Wolyniec P, Pulver AE, Valle D. Fine mapping on chromosome 10q22-q23 implicates Neuregulin 3 in schizophrenia. Am J Hum Genet. 2009;84:21-34. Abstract

Clarke GM, Cardon LR. Aspects of observing and claiming allele flips in association studies. Genet Epidemiol. 2010;34:266-74. Abstract

Fallin MD, Lasseter VK, Wolyniec PS, McGrath JA, Nestadt G, Valle D, Liang KY, Pulver AE. Genomewide linkage scan for schizophrenia susceptibility loci among Ashkenazi Jewish families shows evidence of linkage on chromosome 10q22. Am J Hum Genet. 2003;73:601-11. Abstract

Kao WT, Wang Y, Kleinman JE, Lipska BK, Hyde TM, Weinberger DR, Law AJ. Common genetic variation in Neuregulin 3 (NRG3) influences risk for schizophrenia and impacts NRG3 expression in human brain. Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15619-24. Abstract

Morar B, Dragovic M, Waters FAV, Chandler D, Kalaydjieva L, Jablensky A. Neuregulin 3 (NRG3) as a susceptibility gene in a schizophrenia subtype with florid delusions and relatively spared cognition. Mol Psychiatry. 2010 June 15. Abstract

Wang YC, Chen JY, Chen ML, Chen CH, Lai IC, Chen TT, Hong CJ, Tsai SJ, Liou YL. Neuregulin 3 genetic variations and susceptibility to schizophrenia in a Chinese population. Biol Psychiatry. 2008;64:1093-6. Abstract

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Related News: Neuregulin and ErbB4: Synaptic Jacks of All Trades

Comment by:  Michael CahillPeter Penzes
Submitted 29 April 2011
Posted 29 April 2011
  I recommend the Primary Papers

The recent study by Pitcher et al. provides a novel mechanism linking NRG1/ErbB4 activity to the suppression of NMDAR activity in a manner requiring Src kinase inhibition. The study uses biochemical manipulation of Src activation, as well as studies on cells lacking Src, to examine the role for Src kinase on the effects of NRG1 on NMDAR responses in pyramidal neurons. Overall, the study provides convincing evidence indicating that Src inhibition by NRG1 is an important contributor to the effects of NRG1 on NMDAR pyramidal neuronal hypofunction. The effect of NRG1 and ErbB4 on Src family kinase activation remains complex. Previous studies have found that NRG1 can activate Src, and that inhibition of Src family kinases can block some of the effects of NRG1 on cells, including cellular migration and proliferation (Eckert et al., 2009; Grossmann et al., 2009). Moreover, ErbB4 activity is able to activate fyn when overexpressed in heterologous cells, and NRG1 treatment activates fyn in cells expressing endogenous ErbB4 (Bjarnadottir et al., 2007). Recent findings have similarly found that ErbB4 can activate Src kinases in heterologous cells and indicate that Src family kinase activation, particularly that of fyn, has a role in regulating the effects of NRG1 on interneuron morphology through RhoGEF activity (Cahill et al., 2011).

The findings of Picher et al. indicating that NRG1 can suppress Src kinase are not incompatible with these previously discussed studies, however. Indeed, studies have found that Src kinases can be both activated and inhibited by NRG1 treatment in a cyclic manner (Eckert et al., 2009), suggesting that the duration of NRG1 activity is an important consideration. The effects of NRG1 on pyramidal neuronal structure and/or function also seem to differ depending on the length of NRG1 treatment, as studies have found that chronic NRG1 or ErbB4 activity can promote synaptic structure and/or function (e.g., Barros et al., 2010; Li et al., 2007), whereas short-term NRG1 treatment is detrimental to pyramidal neuronal function (e.g., Wen et al., 2010), indicative of the importance of treatment duration to the functional consequences on neurons. The location of the examined effects is also an important consideration, as biochemical and morphological effects in pyramidal neurons and interneurons might differ following NRG1 treatment, potentially due to differences in ErbB4 expression profiles in these cells (Vullhorst et al., 2009). Given the links of NRG1/ErbB4 to schizophrenia, understanding how short-term and long-term activity of these molecules regulates both interneuron and pyramidal neuron function is of special importance, and merits further studies.

References:

Eckert JM, Byer SJ, Clodfelder-Miller BJ, Carroll SL. Neuregulin-1 beta and neuregulin-1 alpha differentially affect the migration and invasion of malignant peripheral nerve sheath tumor cells. Glia . 2009 Nov 1 ; 57(14):1501-20. Abstract

Grossmann KS, Wende H, Paul FE, Cheret C, Garratt AN, Zurborg S, Feinberg K, Besser D, Schulz H, Peles E, Selbach M, Birchmeier W, Birchmeier C. The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development. Proc Natl Acad Sci U S A . 2009 Sep 29 ; 106(39):16704-9. Abstract

Bjarnadottir M, Misner DL, Haverfield-Gross S, Bruun S, Helgason VG, Stefansson H, Sigmundsson A, Firth DR, Nielsen B, Stefansdottir R, Novak TJ, Stefansson K, Gurney ME, Andresson T. Neuregulin1 (NRG1) signaling through Fyn modulates NMDA receptor phosphorylation: differential synaptic function in NRG1+/- knock-outs compared with wild-type mice. J Neurosci . 2007 Apr 25 ; 27(17):4519-29. Abstract

Cahill ME, Jones KA, Rafalovich I, Xie Z, Barros CS, Müller U, Penzes P. Control of interneuron dendritic growth through NRG1/erbB4-mediated kalirin-7 disinhibition. Mol Psychiatry . 2011 Apr 12. Abstract

Eckert JM, Byer SJ, Clodfelder-Miller BJ, Carroll SL. Neuregulin-1 beta and neuregulin-1 alpha differentially affect the migration and invasion of malignant peripheral nerve sheath tumor cells. Glia . 2009 Nov 1 ; 57(14):1501-20. Abstract

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. Proc Natl Acad Sci U S A . 2009 Mar 17 ; 106(11):4507-12. Abstract

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

Wen L, Lu YS, Zhu XH, Li XM, Woo RS, Chen YJ, Yin DM, Lai C, Terry AV, Vazdarjanova A, Xiong WC, Mei L. Neuregulin 1 regulates pyramidal neuron activity via ErbB4 in parvalbumin-positive interneurons. Proc Natl Acad Sci U S A . 2010 Jan 19 ; 107(3):1211-6. Abstract

Vullhorst D, Neddens J, Karavanova I, Tricoire L, Petralia RS, McBain CJ, Buonanno A. Selective expression of ErbB4 in interneurons, but not pyramidal cells, of the rodent hippocampus. J Neurosci . 2009 Sep 30 ; 29(39):12255-64. Abstract

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Related News: ErbB4 Deletion Models Aspects of Schizophrenia

Comment by:  Beatriz RicoOscar Marin
Submitted 30 October 2013
Posted 5 November 2013

We would like to provide an answer to the question raised by Andrés Buonanno: “If the knockouts have more γ power, why do they perform less well on the Y maze?” As explained in the manuscript, the abnormal increase in γ power observed in conditional ErbB4 mutants would not necessarily lead to better performance, because interneurons are not pacing pyramidal cells at the proper/normal rhythm. In addition, local hypersynchrony seems to affect long-range functional connectivity: We showed a prominent decoupling between the hippocampus and prefrontal cortex. The increase in excitability and synchrony, and the decoupling between the hippocampus and prefrontal cortex, are likely the cause of the behavioral deficits in cognitive function.

In line with this, we respectfully disagree with Buonanno's next comment that “these data are also at odds with what has been observed in schizophrenia.” Indeed, as we mentioned in the manuscript, recent studies indicate that medication-naive, first-episode, and chronic patients with schizophrenia show elevated γ-band power in resting state. Baseline increases in γ oscillations are consistent with increases in the excitatory/inhibitory ratio of cortical neurons. Thus, cortical rhythm abnormalities in schizophrenia seem to include both abnormal increases in baseline power—as we observed in conditional ErbB4 mutants—as well as deficits in task-related oscillations (Uhlhaas and Singer, 2012).

References:

Uhlhaas PJ, and Singer W. (2012). Neuronal dynamics and neuropsychiatric disorders: toward a translational paradigm for dysfunctional large-scale net- works. Neuron 75, 963–980. Abstract

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