A key component in the response of the nervous system to injury is the proliferation and switch to a "proinflammatory" phenotype by microglia (microgliosis). In situations where the blood-brain barrier is intact, microglial numbers increase via the proliferation and chemotaxis of resident microglia; however, there is limited knowledge regarding the factors mediating this response. After peripheral nerve injury, a dorsal horn microgliosis develops, which directly contributes to the development of neuropathic pain. Neuregulin-1 (NRG-1) is a growth and differentiation factor with a well characterized role in neural and cardiac development. Microglia express the NRG1 receptors erbB2, 3, and 4, and NRG1 signaling via the erbB2 receptor stimulated microglial proliferation, chemotaxis, and survival, as well as interleukin-1 release in vitro. Intrathecal treatment with NRG1 resulted in microglial proliferation within the dorsal horn, and these cells developed an activated morphology. This microglial response was associated with the development of both mechanical and cold pain-related hypersensitivity. Primary afferents express NRG1, and after spinal nerve ligation (SNL) we observed both an increase in NRG1 within the dorsal horn as well as activation of erbB2 specifically within microglia. Blockade of the erbB2 receptor or sequestration of endogenous NRG after SNL reduced the proliferation, the number of microglia with an activated morphology, and the expression of phospho-P38 by microglia. Furthermore, consequent to such changes, the mechanical pain-related hypersensitivity and cold allodynia were reduced. NRG1-erbB signaling therefore represents a novel pathway regulating the injury response of microglia.
To determine the possible role of the epigenetic mechanisms in carcinogenesis of the hepatocellular carcinoma, we methylation-profiled the promoter CpG islands of twenty four genes both in HCC tumors and the neighboring non-cancerous tissues of twenty eight patients using the methylation-specific PCR (MSP) method in conjunction with the DNA sequencing. In comparison with the normal liver tissues from the healthy donors, it was found that while remained unmethylated the ABL, CAV, EPO, GATA3, LKB1, NEP, NFL, NIS and p27KIP1 genes, varying extents of the HCC specific hypermethylation were found associated with the ABO, AR, CSPG2, cyclin a1, DBCCR1, GALR2, IRF7, MGMT, MT1A, MYOD1, OCT6, p57 KIP2 , p73, WT1 genes, and demethylation with the MAGEA1 gene, respectively. Judged by whether the hypermethylated occurred in HCC more frequently than in their neighboring normal tissues, the hypermethylation status of the AR, DBCCR1, IRF7, OCT6, and p73 genes was considered as the event specific to the late stage, while that the rest that lacked such a distinguished contrast, as the event specific to the early stage of HCC carcinogenesis. Among all the clinical pathological parameters tested for the association with, the hypermethylation of the cyclin a1 gene was more prevalent in the non-cirrhosis group (P=0.021) while the hypermethylated p16INK4a gene was more common in the cirrhosis group (P=0.017). The concordant methylation behaviors of nineteen genes, including the four previously studied and their association with cirrhosis has been evaluated by the best subgroup selection method. The data presented in this report would enable us to shape our understanding of the mechanisms for the HCC specific loss of the epigenetic stability of the genome, as well as the strategy of developing the novel robust methylation based diagnostic and prognostic tools.
The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development.
Following peripheral nerve injury microglia accumulate within the spinal cord and adopt a proinflammatory phenotype a process which contributes to the development of neuropathic pain. We have recently shown that neuregulin-1, a growth factor released following nerve injury, activates erbB 2, 3, and 4 receptors on microglia and stimulates proliferation, survival and chemotaxis of these cells. Here we studied the intracellular signaling pathways downstream of neuregulin-1-erbB activation in microglial cells. We found that neuregulin-1 in vitro induced phosphorylation of ERK1/2 and Akt without activating p38MAPK. Using specific kinase inhibitors we found that the mitogenic effect of neuregulin-1 on microglia was dependant on MEK/ERK1/2 pathway, the chemotactic effect was dependant on PI3K/Akt signaling and survival was dependant on both pathways. Intrathecal treatment with neuregulin-1 was associated with microgliosis and development of mechanical and cold pain related hypersensitivity which was dependant on ERK1/2 phosphorylation in microglia. Spinal nerve ligation results in a robust microgliosis and sustained ERK1/2 phosphorylation within these cells. This pathway is downstream of neuregulin-1/erbB signaling since its blockade resulted in a significant reduction in microglial ERK1/2 phosphorylation. Inhibition of the MEK/ERK1/2 pathway resulted in decreased spinal microgliosis and in reduced mechanical and cold hypersensitivity after peripheral nerve damage. We conclude that neuregulin-1 released after nerve injury activates microglial erbB receptors which consequently stimulates the MEK/ERK1/2 pathway that drives microglial proliferation and contributes to the development of neuropathic pain. © 2011 Wiley-Liss, Inc.
Adult neurogenesis persists in the rodent dentate gyrus and is stimulated by chronic treatment with conventional antidepressants through BDNF/TrkB signaling. Ketamine in low doses produces both rapid and sustained antidepressant effects in patients. Previous studies have shed light on post-transcriptional synaptic NMDAR mediated mechanisms underlying the acute effect, but how ketamine acts at the cellular level to sustain this anti-depressive function for prolonged periods remains unclear. Here we report that ketamine accelerates differentiation of doublecortin-positive adult hippocampal neural progenitors into functionally mature neurons. This process requires TrkB-dependent ERK pathway activation. Genetic ablation of TrkB in neural stem/progenitor cells, or pharmacologic disruption of ERK signaling, or inhibition of adult neurogenesis, each blocks the ketamine-induced behavioral responses. Conversely, enhanced ERK activity via Nf1 gene deletion extends the response and rescues both neurogenic and behavioral deficits in mice lacking TrkB. Thus, TrkB-dependent neuronal differentiation is involved in the sustained antidepressant effects of ketamine.
During peripheral nervous system development, successful communication between axons and Schwann cells is required for proper function of both myelinated and non-myelinated nerve fibers. Alternatively-spliced proteins belonging to the neuregulin1 (NRG1) gene family of growth and differentiation factors are essential for Schwann cell survival and peripheral nerve development. While recent studies have strongly implicated membrane-bound NRG1 forms (type III) in the myelination at late stages, little is known about the role of soluble, heparin-binding forms of NRG1 (type I/II) in regulating early Schwann cell development in vivo. These forms are rapidly released from axons in vitro by Schwann cell-secreted neurotrophic factors, and, unlike membrane-bound forms, have a unique ability to diffuse and adhere to heparan sulfate-rich cell surfaces. Here, we show that axon-derived soluble NRG1 translocates from axonal to Schwann cell surfaces in the embryonic chick between days 5–7, corresponding to the critical period of Schwann cell survival. Down-regulating endogenous type I/II NRG1 signaling either with a targeted NRG1 antagonist or by shRNA, blocks their differentiation from precursors into immature Schwann cells and increases programmed cell death, while up-regulating NRG1 rescues Schwann cells. Exogenous BDNF also promotes Schwann cell survival through promoting the local release of axonal NRG1. Consistently, increased Schwann cell death occurs both in trkB knock-out mice and after knocking-down axonal trkB in chick embryos, which can then be rescued with soluble NRG1. These findings suggest a localized, axoglial feedback loop through soluble NRG1 and BDNF critical for early Schwann cell survival and differentiation.
Neuregulin (NRG)2 has been shown to have a wide array of functions with important roles in nervous system and heart development as well as in diseases ranging from breast cancer to schizophrenia (1-4). It mediates its diverse effects through binding homo-and heterodimeric cell-surface epidermal growth factor receptors, including HER2(erbB2), HER3(erbB3), and HER4-(erbB4), that leads to rapid receptor-tyrosine phosphorylation and activation of downstream signaling pathways, including the mitogen-activated protein kinase and phosphatidylinositol 3-kinase (5). Many alternatively spliced forms of NRG are secreted after proteolytic cleavage from their transmembrane precursors (6). All of these forms have a receptor-binding epidermal growth factor-like domain together with a distinct C2 immunoglobulin-like domain that functions as a unique heparin-binding domain (HBD) with an alternating array of positively charged amino acids held together in a disulfide-bonded loop. This HBD binds specific sulfate groups on negatively charged heparan sulfate proteoglycans that leads to a highly specific tissue distribution of NRG (7-10).Because of its strong mitogenic effect in some breast cancers, blocking NRG signaling has become an attractive therapeutic target (11). Although a currently approved humanized monoclonal antibody against HER2 called trastuzumab (12) has become a clinically effective adjuvant therapy for a subgroup of breast cancer patients (13, 14), it does not effectively block HER2 activation induced by NRG autocrine and paracrine signaling (15-17). A major obstacle in blocking NRG signaling both in cancer and in developmental studies comes from its ability to become highly concentrated in the extracellular matrix where it can produce sustained HER activation required for downstream gene activations (7, 15). To overcome this obstacle, we have fused the human the HBD of NRG to the soluble ectodomain of HER4 (H4) with high affinity for epidermal growth factor-like domain as a means to target this NRG antagonist to the same heparan sulfate-rich cell surfaces that bind NRG. In this way, the antagonist could effectively compete with NRG on an even footing and would enable more effective studies of the role of NRG in development and in cancer. EXPERIMENTAL PROCEDURESConstruction of Fusion Proteins-All fusion proteins were derived entirely from human sequences. The extracellular domain of HER4 receptor (H4) corresponds to 99 -2042 bp of the human HER4 NM_005235 mRNA. The sequence from 99 -173 bp encodes a 25-amino acid signal sequence that was incorporated onto the N terminus of all constructs for secretion and protein expression. H4 was amplified by PCR and then inserted into pMH vector (Roche Applied Science, Indianapolis IN) between KpnI and EcoRI to generate the H4-HA construct. The HBD (532-849 bp) and HBD-S (532-1023 bp) domains of NRG 1 form (NM_013964) were amplified from the plasmid HARIA PATH 2 (gift of Dr. Tejvir Khurana, University of Pennsylvania). Either HBD-S or the HBD domain alone was inserted into pMH-H...
dStudies on large double-stranded DNA (dsDNA) viruses such as poxviruses have been helpful in identifying a number of viral and cellular growth factors that contribute to our broad understanding of virus-host interaction. Orthopoxviruses and leporipoxviruses are among the most studied viruses in this aspect. However, tanapoxvirus (TPV), a member of the genus Yatapoxvirus, still remains largely unexplored, as the only known hosts for this virus are humans and monkeys. Here, we describe the initial characterization of an epidermal growth factor (EGF)-like growth factor mimicking human neuregulin from TPV, expressed by the TPV-15L gene. Assays using a baculovirus-expressed and tagged TPV-15L protein demonstrated the ability to phosphorylate neuregulin receptors. Neuregulins represent a large family of EGF-like growth factors that play important roles in embryonic endocardium development, Schwann and oligodendrocyte survival and differentiation, localized acetylcholine receptor expression at the neuromuscular junction, and epithelial morphogenesis. Interestingly, certain neuregulin molecules are able to target specific tissues through interactions with heparin sulfate proteoglycans via an immunoglobulin (Ig)-like domain. Analyses of TPV-15L revealed no Ig-like domain, but it retains the ability to bind heparin and phosphorylate neuregulin receptors, providing compelling evidence that TPV-15L is a functional mimetic of neuregulin. TPV-15L knockout virus experiments demonstrate that the virus replicates in human umbilical vein endothelial cells less efficiently than wild-type TPV-Kenya, indicating that this is a nonessential protein for virus viability but can serve a stimulatory role for replication in some cultured cells. However, the precise role of this protein in host-virus interaction still remains to be deduced.
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