Probes for NRG1

Numerous genetic and functional studies implicate variants of Neuregulin-1 (NRG1) and its neuronal receptor ErbB4 in schizophrenia and many of its endophenotypes. Although the neurophysiological and behavioral phenotypes of NRG1 mutant mice have been investigated extensively, practically nothing is known about the function of NRG2, the closest NRG1 homolog. We found that NRG2 expression in the adult rodent brain does not overlap with NRG1 and is more extensive than originally reported, including expression in the striatum and medial prefrontal cortex (mPFC), and therefore generated NRG2 knockout mice (KO) to study its function. NRG2 KOs have higher extracellular dopamine levels in the dorsal striatum but lower levels in the mPFC; a pattern with similarities to dopamine dysbalance in schizophrenia. Like ErbB4 KO mice, NRG2 KOs performed abnormally in a battery of behavioral tasks relevant to psychiatric disorders. NRG2 KOs exhibit hyperactivity in a novelty-induced open field, deficits in prepulse inhibition, hypersensitivity to amphetamine, antisocial behaviors, reduced anxiety-like behavior in the elevated plus maze and deficits in the T-maze alteration reward test-a task dependent on hippocampal and mPFC function. Acute administration of clozapine rapidly increased extracellular dopamine levels in the mPFC and improved alternation T-maze performance. Similar to mice treated chronically with N-methyl-d-aspartate receptor (NMDAR) antagonists, we demonstrate that NMDAR synaptic currents in NRG2 KOs are augmented at hippocampal glutamatergic synapses and are more sensitive to ifenprodil, indicating an increased contribution of GluN2B-containing NMDARs. Our findings reveal a novel role for NRG2 in the modulation of behaviors with relevance to psychiatric disorders.

The Neuregulin (NRG) family of ErbB ligands is comprised of numerous variants originating from the use of different genes, alternative promoters and splice variants. NRGs have generally been thought to be transported to axons and presynaptic terminals where they signal via ErbB3/4 receptors in paracrine or juxtacrine mode. However, we recently demonstrated that unprocessed pro-NRG2 accumulates on cell bodies and proximal dendrites, and that NMDAR activity is required for shedding of its ectodomain by metalloproteinases. Here we systematically investigated the subcellular distribution and processing of major NRG isoforms in rat hippocampal neurons. We show that NRG1 isotypes I and II, which like NRG2 are single-pass transmembrane proteins with an Ig-like domain, share the same subcellular distribution and ectodomain shedding properties. We furthermore show that NRG3, like CRD-NRG1, is a dual-pass transmembrane protein that harbors a second transmembrane domain near its amino-terminus. Both NRG3 and CRD-NRG1 cluster on axons through juxtacrine interactions with ErbB4 present on GABAergic interneurons. Interestingly, while single-pass NRGs accumulate as unprocessed pro-forms, axonal puncta of CRD-NRG1 and NRG3 are comprised of processed protein. Mutations of CRD-NRG1 and NRG3 that render them resistant to BACE cleavage, as well as BACE inhibition, result in the loss of axonal puncta and in the accumulation of unprocessed proforms in neuronal soma. Together, these results define two groups of NRGs with distinct membrane topologies and fundamentally different targeting and processing properties in central neurons. The implications of this functional diversity for the regulation of neuronal processes by the NRG/ErbB pathway are discussed.SIGNIFICANCE STATEMENTNumerous Neuregulins are generated through the use of different genes, promoters and alternative splicing, but the functional significance of this evolutionary conserved diversity remains poorly understood. Here we show that NRGs can be categorized by their membrane topologies. Single-pass Neuregulins such as NRG1 types I/II and NRG2 accumulate as unprocessed pro-forms on cell bodies, and their ectodomains are shed by metalloproteinases in response to NMDA receptor activation. By contrast, dual-pass CRD-NRG1 and NRG3 are constitutively processed by BACE and accumulate on axons where they interact with ErbB4 in juxtacrine mode. These findings reveal a previously unknown functional relationship between membrane topology, protein processing and subcellular distribution, and suggest that single- and dual-pass NRGs regulate neuronal functions in fundamentally different ways.

In contrast to acute peripheral nerve injury, the molecular response of Schwann cells in chronic neuropathies remains poorly understood. Onion bulb structures are a pathological hallmark of demyelinating neuropathies, but the nature of these formations is unknown. Here, we show that Schwann cells induce the expression of Neuregulin-1 type I (NRG1-I), a paracrine growth factor, in various chronic demyelinating diseases. Genetic disruption of Schwann cell-derived NRG1 signalling in a mouse model of Charcot-Marie-Tooth Disease 1A (CMT1A), suppresses hypermyelination and the formation of onion bulbs. Transgenic overexpression of NRG1-I in Schwann cells on a wildtype background is sufficient to mediate an interaction between Schwann cells via an ErbB2 receptor-MEK/ERK signaling axis, which causes onion bulb formations and results in a peripheral neuropathy reminiscent of CMT1A. We suggest that diseased Schwann cells mount a regeneration program that is beneficial in acute nerve injury, but that overstimulation of Schwann cells in chronic neuropathies is detrimental.

Gene expression analysis is essential for understanding the rich repertoire of cellular functions. With the development of sensitive molecular tools such as single-cell RNA sequencing, extensive gene expression data can be obtained and analyzed from various tissues. Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful complementary tool for single-cell genomics studies because of its ability to map and quantify the spatial distributions of single mRNAs at the subcellular level in their native tissue. Here, we present a detailed method to study the copy numbers and spatial localizations of single mRNAs in the cochlea and inferior colliculus. First, we demonstrate that smFISH can be performed successfully in adult cochlear tissue after decalcification. Second, we show that the smFISH signals can be detected with high specificity. Third, we adapt an automated transcript analysis pipeline to quantify and identify single mRNAs in a cell-specific manner. Lastly, we show that our method can be used to study possible correlations between transcriptional and translational activities of single genes. Thus, we have developed a detailed smFISH protocol that can be used to study the expression of single mRNAs in specific cell types of the peripheral and central auditory systems.

Genetic variants of Neuregulin 1 (NRG1) and its neuronal tyrosine kinase receptor ErbB4 are associated with risk for schizophrenia, a neurodevelopmental disorder characterized by excitatory/inhibitory imbalance and dopamine (DA) dysfunction. To date, most ErbB4 studies have focused on GABAergic interneurons in the hippocampus and neocortex, particularly fast-spiking parvalbumin-positive (PV+) basket cells. However, NRG has also been shown to modulate DA levels, suggesting a role for ErbB4 signaling in dopaminergic neuron function. Here we report that ErbB4 in midbrain DAergic axonal projections regulates extracellular DA levels and relevant behaviors. Mice lacking ErbB4 in tyrosine hydroxylase-positive (TH+) neurons, but not in PV+ GABAergic interneurons, exhibit different regional imbalances of basal DA levels and fail to increase DA in response to local NRG1 infusion into the dorsal hippocampus, medial prefrontal cortex and dorsal striatum measured by reverse microdialysis. Using Lund Human Mesencephalic (LUHMES) cells, we show that NRG/ErbB signaling increases extracellular DA levels, at least in part, by reducing DA transporter (DAT)-dependent uptake. Interestingly, TH-Cre;ErbB4f/f mice manifest deficits in learning, spatial and working memory-related behaviors, but not in numerous other behaviors altered in PV-Cre;ErbB4f/fmice. Importantly, microinjection of a Cre-inducible ErbB4 virus (AAV-ErbB4.DIO) into the mesencephalon of TH-Cre;ErbB4f/f mice, which selectively restores ErbB4 expression in DAergic neurons, rescues DA dysfunction and ameliorates behavioral deficits. Our results indicate that direct NRG/ErbB4 signaling in DAergic axonal projections modulates DA homeostasis, and that NRG/ErbB4 signaling in both GABAergic interneurons and DA neurons contribute to the modulation of behaviors relevant to psychiatric disorders.