ErbB4 signaling in dopaminergic axonal projections increases extracellular dopamine levels and regulates spatial/working memory behaviors.

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/f mice. 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.

Neuregulin-2 ablation results in dopamine dysregulation and severe behavioral phenotypes relevant to psychiatric disorders.

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.

Regulation of ErbB-4 endocytosis by neuregulin in GABAergic hippocampal interneurons.

Neuregulin (NRG)/ErbB receptor signaling pathways have recently been implicated in the reversal of long-term potentiation at hippocampal glutamatergic synapses. Moreover, polymorphisms in NRG-1 and ErbB-4 genes have been linked to an increased risk for developing schizophrenia. ErbB-4 is highly expressed at glutamatergic synapses where it binds to PSD-95 via its carboxyl terminal T-V-V sequence. Here we investigated the expression, localization and trafficking of ErbB-4 in cultured hippocampal neurons by immunocytochemistry, surface protein biotinylation, and live labeling of native receptors. We show that neuronal ErbB-4 is detected at its highest levels in GABAergic interneurons, as observed in vivo. ErbB-4 immunoreactivity precedes PSD-95 expression, with ErbB-4 cluster initially forming in the absence of, but later associating with, PSD-95-positive puncta. By surface protein biotinylation, the fraction of ErbB-4 receptors on the plasma membrane increases from 30% to 65% between 6 and 16 days in vitro (DIV). Interestingly, 30 min of NRG stimulation triggers measurable ErbB-4 receptor internalization at DIV 16, despite increased colocalization with PSD-95. We also investigated the role of TNFalpha-converting enzyme (TACE)-mediated receptor processing in regulating ErbB-4 surface expression. We found that the cleavage-resistant JM-b isoform accounts for 80% of all ErbB-4 transcripts in cultured hippocampal neurons. Receptor stimulation or treatment with phorbol esters does not induce detectable ErbB-4 processing, indicating that neurons mostly rely on endocytosis of the intact receptor to regulate ErbB-4 surface expression. These results enhance our understanding of the regulation of ErbB-4--mediated signaling at glutamatergic synapses.

Molecular dissection of DNA sequences and factors involved in slow muscle-specific transcription.

Transcription is a major regulatory mechanism for the generation of slow- and fast-twitch myofibers. We previously identified an upstream region of the slow TnI gene (slow upstream regulatory element [SURE]) and an intronic region of the fast TnI gene (fast intronic regulatory element [FIRE]) that are sufficient to direct fiber type-specific transcription in transgenic mice. Here we demonstrate that the downstream half of TnI SURE, containing E box, NFAT, MEF-2, and CACC motifs, is sufficient to confer pan-skeletal muscle-specific expression in transgenic mice. However, upstream regions of SURE and FIRE are required for slow and fast fiber type specificity, respectively. By adding back upstream SURE sequences to the pan-muscle-specific enhancer, we delineated a 15-bp region necessary for slow muscle specificity. Using this sequence in a yeast one-hybrid screen, we isolated cDNAs for general transcription factor 3 (GTF3)/muscle TFII-I repeat domain-containing protein 1 (MusTRD1). GTF3 is a multidomain nuclear protein related to initiator element-binding transcription factor TF II-I; the genes for both proteins are deleted in persons with Williams-Beuren syndrome, who often manifest muscle weakness. Gel retardation assays revealed that full-length GTF3, as well as its carboxy-terminal half, specifically bind the bicoid-like motif of SURE (GTTAATCCG). GTF3 expression is neither muscle nor fiber type specific. Its levels are highest during a period of fetal development that coincides with the emergence of specific fiber types and transiently increases in regenerating muscles damaged by bupivacaine. We further show that transcription from TnI SURE is repressed by GTF3 when overexpressed in electroporated adult soleus muscles. These results suggest a role for GTF3 as a regulator of slow TnI expression during early stages of muscle development and suggest how it could contribute to Williams-Beuren syndrome.

The genomic basis of K(V)3.4 potassium channel mRNA diversity in mice.

K(V)3.4 belongs to the shaw subfamily of shaker-type potassium channels. It conducts fast inactivating, high threshold currents in the central nervous system and in fast-twitch skeletal muscle fibers. The corresponding mouse gene, Kcnc4, consists of five exons spanning a region of 20 kb. Approximately 700 bp of regulatory sequence were delineated. It is GC-rich and lacks typical TATA and CAAT motifs. Instead, seven Sp-1 and three E-box elements define putative regulatory sequences. The mouse K(V)3.4 mRNA has a size of 3639 bp, 1120 bp of which are 3' untranslated region. A transcript initiated from an alternative 5'-exon was identified by RACE and verified by genomic analysis. This isoform, designated K(V)3.4d, is predominantly expressed in skeletal muscle and probably results from alternative promoter usage. It encodes a channel protein with a novel N-terminal cytoplasmic domain. It lacks the conserved sequence motifs encoding the shaw-type tetramerization domain and the 'ball' peptide, which confers fast inactivation properties. Another splice variant, K(V)3.4c, is derived by exon skipping in the C-terminal region and is expressed at similar levels in brain and muscle. These data demonstrate that differential splicing and alternative transcription start sites are utilised to generate a set of K(V)3.4 variants in skeletal muscle and brain, presumably involved in the regulation of excitability.

In vitro refolding of heterodimeric CapZ expressed in E. coli as inclusion body protein.

CapZ is a heterodimeric Ca(2+)-independent actin binding protein which plays an important role in organizing the actin filament lattice of cross-striated muscle cells. It caps the barbed end of actin filaments and promotes nucleation of actin polymerization, thereby regulating actin filament length. Here we report the expression of the two muscle-specific isoforms alpha2 and beta1, from chicken in Escherichia coli as individual subunits using the pQE60 expression vector and the subsequent renaturation of the functional CapZ heterodimer from inclusion bodies. Optimal renaturation conditions were obtained both by simultaneous refolding of urea-solubilized subunits and by rapid dilution into a buffer containing 20% glycerol, 5 mM EGTA, 2 mM DTT, 1 mM PMSF, and 100 mM Tris, pH 7.4. The refolding mixture was incubated for 24 h at 15 degrees C and the protein was concentrated by ultrafiltration. Biochemical characterization of the recombinant heterodimer revealed actin binding activities indistinguishable from those of native CapZ as purified from chicken skeletal muscle. Using the same protocol, we were able to refold the beta1, but not the alpha2 isoform as a single polypeptide, indicating a role for beta1 as a molecular template for the folding of alpha2. The reported recombinant approach leads to high yields of active heterodimer and allows the renaturation and characterization of the beta subunit.

Expression of the potassium channel KV3.4 in mouse skeletal muscle parallels fiber type maturation and depends on excitation pattern.

We report the detailed expression pattern of the voltage-dependent potassium channel KV3.4 (rat homologue, Raw3) in mouse skeletal muscle. Using semi-quantitative RT-PCR, we show that its expression is detectable at embryonic day 17 and rises to adult levels within 2 weeks after birth. Expression is fiber type-dependent, with mRNA levels being 5-6-fold lower in the mixed slow/fast soleus muscle than in the fast tibialis anterior and extensor digitorum longus muscles. Fast muscles from myotonic mice exhibit low KV3.4 mRNA levels similar to those of wild-type soleus. In denervated extensor digitorum longus, KV3.4 expression declines to perinatal levels. We conclude that KV3.4 expression in mouse skeletal muscle is regulated by the pattern of excitation.

Expression of chloride channel 1 mRNA in cultured myogenic cells: a marker of myotube maturation.

The chloride channel CIC-1 is required to maintain a normal excitability of mature muscle fibers; its blockade leads to hyperexcitability, the hallmark of the disease myotonia. In mouse and rat myotubes, representing the embryonic stage of muscle, CIC-1 mRNA is not detectable by Northern blotting. From neonatal to adult, CIC-1 expression increases at least fourfold. Using RT-PCR and hybridization on cultured myotubes we found CIC-1 mRNA at a level of 0.4-1.1% of that in mature mouse muscle, and < or = 0.01% in myoblasts, at stages when desmin mRNA levels are already high. The level of CIC-1 mRNA is thus a sensitive and specific indicator of the maturation of skeletal muscle cells.