Neuregulin-4 Is Required for Maintaining Soma Size of Pyramidal Neurons in the Motor Cortex.

The regulation of neuronal soma size is essential for appropriate brain circuit function and its dysregulation is associated with several neurodevelopmental disorders. A defect in the dendritic growth and elaboration of motor neocortical pyramidal neurons in neonates lacking neuregulin-4 (NRG4) has previously been reported. In this study, we investigated whether the loss of NRG4 causes further morphologic defects that are specific to these neurons. We analyzed the soma size of pyramidal neurons of layer (L)2/3 and L5 of the motor cortex and a subpopulation of multipolar interneurons in this neocortical region in Nrg4+/+ and Nrg4-/- mice. There were significant decreases in pyramidal neuron soma size in Nrg4-/- mice compared with Nrg4+/+ littermates at all stages studied [postnatal day (P)10, P30, and P60]. The reduction was especially marked at P10 and in L5 pyramidal neurons. Soma size was not significantly different for multipolar interneurons at any age. This in vivo phenotype was replicated in pyramidal neurons cultured from Nrg4-/- mice and was rescued by NRG treatment. Analysis of a public single-cell RNA sequencing repository revealed discrete Nrg4 and Erbb4 expression in subpopulations of L5 pyramidal neurons, suggesting that the observed defects were due in part to loss of autocrine Nrg4/ErbB4 signaling. The pyramidal phenotype in the motor cortex of Nrg4-/- mice was associated with a lack of Rotarod test improvement in P60 mice, suggesting that absence of NRG4 causes alterations in motor performance.

Neuregulin-4 Is Required for the Growth and Elaboration of Striatal Medium Spiny Neuron Dendrites.

Medium spiny neurons (MSNs) comprise the vast majority of neurons in the striatum. Changes in the exuberant dendrites of these widely connected neurons are associated with a multitude of neurological conditions and are caused by a variety of recreational and medicinal drugs. However, we have a poor understanding of the physiological regulators of dendrite growth and elaboration of this clinically important population of neurons. Here, we show that MSN dendrites are markedly smaller and less branched in neonatal mice that possess a homozygous null mutation in the neuregulin-4 gene (Nrg4-/-) compared with wild type (Nrg4+/+) littermates. Nrg4-/- mice also had a highly significant reduction in MSN dendrite spine number in neonates and adults. The striking stunted dendrite arbor phenotype of MSNs observed in Nrg4-/- neonates was replicated in MSNs cultured from Nrg4-/- embryos and was completely rescued by soluble recombinant neuregulin-4. MSNs cultured from wild type mice coexpressed NRG4 and its receptor ErbB4. Our findings show that NRG4 is a major novel regulator of dendritic growth and arborization and spine formation in the striatum and suggest that it exerts its effects by an autocrine/paracrine mechanism.

An essential role for neuregulin-4 in the growth and elaboration of developing neocortical pyramidal dendrites.

Neuregulins, with the exception of neuregulin-4 (NRG4), have been shown to be extensively involved in many aspects of neural development and function and are implicated in several neurological disorders, including schizophrenia, depression and bipolar disorder. Here we provide the first evidence that NRG4 has a crucial function in the developing brain. We show that both the apical and basal dendrites of neocortical pyramidal neurons are markedly stunted in Nrg4-/- neonates in vivo compared with Nrg4+/+ littermates. Neocortical pyramidal neurons cultured from Nrg4-/- embryos had significantly shorter and less branched neurites than those cultured from Nrg4+/+ littermates. Recombinant NRG4 rescued the stunted phenotype of embryonic neocortical pyramidal neurons cultured from Nrg4-/- mice. The majority of cultured wild type embryonic cortical pyramidal neurons co-expressed NRG4 and its receptor ErbB4. The difference between neocortical pyramidal dendrites of Nrg4-/- and Nrg4+/+ mice was less pronounced, though still significant, in juvenile mice. However, by adult stages, the pyramidal dendrite arbors of Nrg4-/- and Nrg4+/+ mice were similar, suggesting that compensatory changes in Nrg4-/- mice occur with age. Our findings show that NRG4 is a major novel regulator of dendritic arborisation in the developing cerebral cortex and suggest that it exerts its effects by an autocrine/paracrine mechanism.

Calpain activation induced by glucose deprivation is mediated by oxidative stress and contributes to neuronal damage.

The mechanisms leading to neuronal death during glucose deprivation have not been fully elucidated, but a role of oxidative stress has been suggested. In the present study we have investigated whether the production of reactive oxygen species during glucose deprivation, contributes to the activation of calpain, a calcium-dependent protease involved in neuronal injury associated with brain ischemia and cerebral trauma. We have observed a rapid activation of calpain, as monitored by the cleavage of the cytoskeletal protein α-spectrin, after glucose withdrawal, which is reduced by inhibitors of xanthine oxidase, phospholipase A2 and NADPH oxidase. Results suggest that phospholipase A2 and NADPH oxidase contribute to the early activation of calpain after glucose deprivation. In particular NOX2, a member of the NADPH oxidase family is involved, since reduced stimulation of calpain activity is observed after glucose deprivation in hippocampal slices from transgenic mice lacking a functional NOX2. We observed an additive effect of the inhibitors of xanthine oxidase and phospholipase A2 on both ROS production and calpain activity, suggesting a synergistic action of these two enzymes. The present results provide new evidence showing that reactive oxygen species stimulate calpain activation during glucose deprivation and that this mechanism is involved in neuronal death.

The extracellular-regulated protein kinase 5 (ERK5) promotes cell proliferation through the down-regulation of inhibitors of cyclin dependent protein kinases (CDKs).

Activation of the extracellular-regulated protein kinase 5 (ERK5) has been associated with mitogenic signal transduction. However, conflicting findings have challenged the idea that ERK5 is a critical regulator of cell proliferation. We have addressed this issue by testing the effect of the conditional loss of ERK5 in primary fibroblasts. We have discovered that ERK5 suppressed the expression of the cyclin dependent protein kinase (CDKs) inhibitors, p21 and p27, by decreasing mRNA and protein stability, respectively. As a result, low level CDK2 activity detected in ERK5-deficient cells correlated with a defect in G1 to S phase transition of the cell cycle. Similarly, we found that the malignant MDA-MB-231 human breast cancer cell line was dependent on ERK5 to proliferate. We propose that ERK5 blocks p21 expression in MDA-MB-231 cells via a mechanism that implicates c-Myc-dependent transcriptional regulation of the miR-17-92 cluster. Together with evidence that cancer patients with poor prognosis display a high level of expression of components of the ERK5 signaling pathway, these findings support the hypothesis that ERK5 can be a potential target for cancer therapy.

Activation of NOX2 by the stimulation of ionotropic and metabotropic glutamate receptors contributes to glutamate neurotoxicity in vivo through the production of reactive oxygen species and calpain activation.

Prolonged activation of glutamate receptors leads to excitotoxicity. Several processes such as reactive oxygen species (ROS) production and activation of the calcium-dependent protease, calpain, contribute to glutamate-induced damage. It has been suggested that the ROS-producing enzyme, NADPH oxidase (NOX), plays a role in excitotoxicity. Studies have reported NOX activation after NMDA receptor stimulation during excitotoxic damage, but the role of non-NMDA and metabotropic receptors is unknown. We evaluated the roles of different glutamate receptor subtypes on NOX activation and neuronal death induced by the intrastriatal administration of glutamate in mice. In wild-type mice, NOX2 immunoreactivity in neurons and microglia was stimulated by glutamate administration, and it progressively increased as microglia became activated; calpain activity was also induced. By contrast, mice lacking NOX2 were less vulnerable to excitotoxicity, and there was reduced ROS production and protein nitrosylation, microglial reactivity, and calpain activation. These results suggest that NOX2 is stimulated by glutamate in neurons and reactive microglia through the activation of ionotropic and metabotropic receptors. Neuronal damage involves ROS production by NOX2, which, in turn, contributes to calpain activation.