Susan O. Meakin

Cyclic phosphatidic acid elicits neurotrophin-like actions in embryonic hippocampal neurons.

Cyclic phosphatidic acid (cPA; 1‐acyl‐sn‐glycerol‐2,3‐cyclic phosphate) is an analog of the growth factor‐like phospholipid mediator lysophosphatidic acid (LPA). As brain tissue is the richest source of cPA we tested its effects on hippocampal neurons from day 16/17 embryonic rat cultured in a serum‐free medium. Nanomolar concentrations of cPA elicited a neurotrophic effect and promoted neurite outgrowth that exceeded that of 50u2003ng/mL nerve growth factor (NGF). Pertussis toxin, the LPA1/LPA3 receptor‐selective antagonist dioctylglycerol pyrophosphate, the myristoylated inhibitory pseudosubstrate peptide of protein kinase A (PKI), Wortmannin and PD98059 abolished the neurite‐promoting effect. cPA elicited a sustained activation of extracellular signal‐related kinases (ERK) 1/2 and Akt. Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, reduced cPA‐induced enhancement of neurite outgrowth. In B5P cells, a clonal cell line of PC12 cells overexpressing tyrosine kinase NGF receptor (TrkA), cPA elicited transphosphorylation of TrkA. cPA‐elicited ERK activation was blocked by K252a and PKI. These results suggest that cPA mimics the effects of, and activates signaling pathways similar to, the neurotrophin NGF in cultured embryonic hippocampal neurons and B5P cells.

Activity-dependent interaction of the intracellular domain of rat TrkA with intermediate filament proteins, the β-6 proteasomal subunit, Ras-GRF1, and the p162 subunit of eIF3

Many responses to nerve growth factor (NGF) are regulated through the receptor tyrosine kinase trkA. To understand more fully the functions of trkA in NGF responsive cells, we have expressed the intracellular domain of rat trkA as a fusion protein with the yeast gal4 transcription factor, and used the fusion protein to probe rat and mouse cDNA libraries by the yeast two-hybrid system. We have identified a direct interaction between the intracellular domain of trkA and two members of the intermediate filament (IF) family of proteins, the guanine-nucleotide exchange protein Ras-GRF1, the p162 subunit of eIF3, and the β-6 proteasome subunit. The interactions are dependent on an active trkA kinase, and RasGRF1, the β-6 proteasomal subunit, and peripherin are directly phosphorylated by trkA. The interaction with trkA is not affected by mutations at either Tyr499 or Tyr794, the two major phosphotyrosine residues essential to the activation and receptor binding of Shc, FRS-2/SNT, and phospholipase Cγ-1, and it is highly specific in vitro for trkA, with little or no binding observed with trkB and/or trkC. The results show that trkA may play a regulatory role in a variety of cellular functions in addition to neuritogenesis, including regulated protein degradation and transcriptional activation.

Chapter 7 Central mechanisms for autonomic dysreflexia after spinal cord injury

Publisher Summary This chapter discusses the central mechanisms for autonomic dysreflexia after spinal cord injury. Mechanisms for autonomic dysreflexia include upregulation of vascular catecholamine receptors, increased neural release of catecholamines, loss of the baroreceptor reflex, and loss of tonic bulbospinal inhibitory input to spinal neurons. Spinal cord injury leads to a host of changes within the injured cord that promote pathological secondary outcomes, such as autonomic dysreflexia. Studies show that neurotrophic factors are useful and probably necessary for the process of regeneration of injured tracts and neurons whereas nerve growth factor (NGF) does not appear to be a good candidate for this task as it clearly contributes to secondary disorders. Likewise, inflammation is a necessary part of healing but, in the central nervous system (CNS), inflammation can cause great damage that is not readily reversed. Therefore, the inflammatory response to cord injury also is an ideal target for manipulation to maintain its useful functions while limiting its capacity to cause destructive or maladaptive responses. Autonomic dysreflexia is one of many secondary consequences of cord injury such as bladder and sexual dysfunction, chronic pain, muscle spasticity. Understanding the mechanisms of autonomic dysreflexia and development of treatments to prevent its development may influence many of these disabling secondary disorders.

Developmental expression patterns of the signaling adapters FRS-2 and FRS-3 during early embryogenesis

Two fibroblast growth factor (FGF) receptor substrates (FRS2 and FRS3) are involved in downstream signaling from activated FGF receptors and neurotrophin-activated Trk receptors. Despite the importance of signaling from these factors in embryogenesis, FRS2 and FRS3 expression patterns during development are unknown. In this study we characterize the expression of FRS2 and FRS3 from E7 to parturition and in adult murine tissues. Both are first detected in whole E8.5 CD1 mouse embryos. FRS2 is detected as early as E7 in the developing syncytiotrophoblast, later in the neural tube (NT) and in many adult and fetal tissues. FRS3 is more restricted in location than FRS2 (fetal NT, heart, stomach, liver and some adult tissues), and is expressed predominantly in the ventricular layer of the developing NT and brains of murine embryos.

ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation

Homophilic binding inu2003trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen‐activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)‐mediated neurite outgrowth in the PC12‐E2 cell line. We found that both FGFR substrate‐2 and Grb2 play important roles in NCAM as well as in FGF2‐stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/Fyn signaling pathway.

Nerve growth factor increases connexin43 phosphorylation and gap junctional intercellular communication

The function of gap junctions is regulated by the phosphorylation state of their connexin subunits. Numerous growth factors are known to regulate connexin phosphorylation; however, the effect of nerve growth factor on gap junction function is not understood. The phosphorylation of connexin subunits is a key event during many aspects of the lifecycle of a connexin, including open/close states, assembly/trafficking, and degradation, and thus affects the functionality of the channel. PC12 cells infected with connexin43 (Cx43) retrovirus were used as a neuronal model to characterize the signal transduction pathways activated by nerve growth factor (NGF) that potentially affect the functional state of Cx43. Immunoblot analysis demonstrated that Cx43 and the mitogen‐activated protein kinase (MAPK), ERK‐1/2, were phosphorylated in response to TrkA activation via NGF and that phosphorylation could be prevented by treatment with the MEK‐1/2 inhibitor U0126. The effects of NGF on gap junction intercellular communication were examined by monitoring fluorescence recovery after photobleaching PC12‐Cx43 cells preloaded with calcein. Fluorescence recovery in the photobleached area increased after NGF treatment and decreased when pretreated with the MEK‐1/2 inhibitor U0126. These data are the first to show a direct signaling link between neurotrophins and the phosphorylation of connexin proteins through the MAPK pathway resulting in increased gap junctional intercellular communication. Neurotrophic regulation of connexin activity provides a novel mechanism of regulating intercellular communication between neurons during nervous system development and repair.

Ectopic expression of the TrkA receptor in adult dopaminergic mesencephalic neurons promotes retrograde axonal NGF transport and NGF-dependent neuroprotection.

A recombinant adeno-associated virus (rAAV) was used to investigate the impact of an ectopic expression of the NGF high-affinity receptor in adult neurons. The rat TrkA cDNA cloned in a pCMX vector was first tagged with a human c-Myc sequence. The resulting vector was shown to encode a functional receptor which promoted the expression of TrkA immunoreactivity upon transfection of 293 fibroblasts or nnr5 cells, a TrkA-defective variant of PC12 cells. These cells also accumulate TrkA transcripts upon transfection and extended neurites in the presence of NGF. Therefore, the TrkA(myc) cassette was inserted into the pSSV9 plasmid. The new vectors shared properties similar to pCMX TrkA(myc) in 293 and nnr5 cells and enabled the preparation of rAAV TrkA(myc) viruses. Unilateral injection of this rAAV into the substantia nigra (SN) resulted in a protracted expression of TrkA (or c-Myc) immunoreactivity in numerous cell bodies, including tyrosine-hydroxylase (TH)-positive dopaminergic neurons. The presence of TrkA receptors in corresponding striatal dopaminergic endings was demonstrated by the advent of a striato-nigral retrograde axonal transport of (125)I-NGF. Likewise, ectopic expression of TrkA in neurons of the parafascicular thalamic nucleus promoted a striatofuge transport of NGF toward this structure. To investigate whether ectopic expression of TrkA in SN neurons may confer neuroprotection, lesions were induced by 6-hydroxydopamine in striata located ipsilateral to the virus injection site. NGF or vehicle were next delivered dorsally to the virus-treated SN for 2 weeks, before sacrifice and processing of brains for TH-immunohistochemistry. NGF treatment, in contrast to treatment with vehicle, significantly enhanced the number of dopaminergic neurons counted in the lesioned SN. These data suggest that ectopic TrkA can mediate the trophic actions of NGF and influence neuronal plasticity in vivo.

Nerve growth factor receptor TrkA, a new receptor in insulin signaling pathway in PC12 cells.

Background: TrkA is a transmembrane receptor tyrosine kinase for nerve growth factor. Results: TrkA forms a molecular complex with insulin receptor and IRS-1 to induce Akt and Erk5 phosphorylation. Conclusion: The NGF-TrkA receptor influences insulin signaling. Significance: The TrkA receptor is involved in insulin signaling, and NGF may regulate neuronal glucose uptake as neurons are insulin-insensitive. TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.

Genomic organization and comparative sequence analysis of the mouse and human FRS2, FRS3 genes.

The signaling adapter proteins FRS2 and FRS3 are implicated in the transmission of extracellular signals from nerve growth factor (NGF) or fibroblast growth factor (FGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. This study presents the genomic sequence and exon-intron organization of the mouse FRS2 and FRS3 loci as well as their evolutionary conservation with their human counterparts. Both FRS2 and FRS3 contain 5 coding exons spanning over 7 kb of genomic sequence with similar exon sizes and organization. Comparative genomic sequence analyses show a highly conserved genomic organization between mouse and human in both FRS2 and FRS3 genes. Non-coding sequences, highly conserved between mouse and human, were identified in the FRS3 introns that may potentially function as regulatory elements. To assay potential differences in their patterns of expression, RT-PCR analysis was used to assay FRS2 and FRS3 expression in the developing embryo and neural tube (NT) during the time of neurogenesis.

Role and expression of FRS2 and FRS3 in prostate cancer

BackgroundFGF receptor substrates (FRS2 and FRS3) are key adaptor proteins that mediate FGF-FGFR signalling in benign as well as malignant tissue. Here we investigated FRS2 and FRS3 as a means of disrupting global FGF signalling in prostate cancer.MethodsFRS2 and FRS3 manipulation was investigated in vitro using over-expression, knockdown and functional assays. FRS2 and FRS3 expression was profiled in cell lines and clinical tumors of different grades.ResultsIn a panel of cell lines we observed ubiquitous FRS2 and FRS3 transcript and protein expression in both benign and malignant cells. We next tested functional redundancy of FRS2 and FRS3 in prostate cancer cells. In DU145 cells, specific FRS2 suppression inhibited FGF induced signalling. This effect was not apparent in cells stably over-expressing FRS3. Indeed FRS3 over-expression resulted in enhanced proliferation (p = 0.005) compared to control cells. Given this functional redundancy, we tested the therapeutic principle of dual targeting of FRS2 and FRS3 in prostate cancer. Co-suppression of FRS2 and FRS3 significantly inhibited ERK activation with a concomitant reduction in cell proliferation (p < 0.05), migration and invasion (p < 0.05). Synchronous knockdown of FRS2 and FRS3 with exposure to cytotoxic irradiation resulted in a significant reduction in prostate cancer cell survival compared to irradiation alone (p < 0.05). Importantly, this synergistic effect was not observed in benign cells. Finally, we investigated expression of FRS2 and FRS3 transcript in a cohort of micro-dissected tumors of different grades as well as by immunohistochemistry in clinical biopsies. Here, we did not observe any difference in expression between benign and malignant biopsies.ConclusionsThese results suggest functional overlap of FRS2 and FRS3 in mediating mitogenic FGF signalling in the prostate. FRS2 and FRS3 are not over-expressed in tumours but targeted dual inhibition may selectively adversely affect malignant but not benign prostate cells.