Neurotrophins regulate development, maintenance, and function of vertebrate nervous systems. Neurotrophins activate two different classes of receptors, the Trk family of receptor tyrosine kinases and p75NTR, a member of the TNF receptor superfamily. Through these, neurotrophins activate many signaling pathways, including those mediated by ras and members of the cdc-42/ras/rho G protein families, and the MAP kinase, PI-3 kinase, and Jun kinase cascades. During development, limiting amounts of neurotrophins function as survival factors to ensure a match between the number of surviving neurons and the requirement for appropriate target innervation. They also regulate cell fate decisions, axon growth, dendrite pruning, the patterning of innervation and the expression of proteins crucial for normal neuronal function, such as neurotransmitters and ion channels. These proteins also regulate many aspects of neural function. In the mature nervous system, they control synaptic function and synaptic plasticity, while continuing to modulate neuronal survival.

Neurotrophins: roles in neuronal development and function.

In the past decade, studies of the human tumour suppressor LKB1 have uncovered a novel signalling pathway that links cell metabolism to growth control and cell polarity. LKB1 encodes a serine-threonine kinase that directly phosphorylates and activates AMPK, a central metabolic sensor. AMPK regulates lipid, cholesterol and glucose metabolism in specialized metabolic tissues, such as liver, muscle and adipose tissue. This function has made AMPK a key therapeutic target in patients with diabetes. The connection of AMPK with several tumour suppressors suggests that therapeutic manipulation of this pathway using established diabetes drugs warrants further investigation in patients with cancer.

The LKB1-AMPK pathway: metabolism and growth control in tumour suppression.

The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs) that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRADα/β and MO25α/β. We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRADα and MO25α, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRADα/β and MO25α/β activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRADα or STRADβ and MO25α or MO25β are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1), but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

/pdf/complexes-between-the-lkb1-tumor-suppressor-strada-b-and-512gnrld5b.pdf

Complexes between the LKB1 tumor suppressor, STRADα/β and MO25α/β are upstream kinases in the AMP-activated protein kinase cascade

The AMP-activated protein kinase (AMPK) system acts as a sensor of cellular energy status that is conserved in all eukaryotic cells. It is activated by increases in the cellular AMP:ATP ratio caused by metabolic stresses that either interfere with ATP production (eg, deprivation for glucose or oxygen) or that accelerate ATP consumption (eg, muscle contraction). Activation in response to increases in AMP involves phosphorylation by an upstream kinase, the tumor suppressor LKB1. In certain cells (eg, neurones, endothelial cells, and lymphocytes), AMPK can also be activated by a Ca(2+)-dependent and AMP-independent process involving phosphorylation by an alternate upstream kinase, CaMKKbeta. Once activated, AMPK switches on catabolic pathways that generate ATP, while switching off ATP-consuming processes such as biosynthesis and cell growth and proliferation. The AMPK complex contains 3 subunits, with the alpha subunit being catalytic, the beta subunit containing a glycogen-sensing domain, and the gamma subunits containing 2 regulatory sites that bind the activating and inhibitory nucleotides AMP and ATP. Although it may have evolved to respond to metabolic stress at the cellular level, hormones and cytokines such as insulin, leptin, and adiponectin can interact with the system, and it now appears to play a key role in maintaining energy balance at the whole body level. The AMPK system may be partly responsible for the health benefits of exercise and is the target for the antidiabetic drug metformin. It is a key player in the development of new treatments for obesity, type 2 diabetes, and the metabolic syndrome.

AMP-Activated Protein Kinase in Metabolic Control and Insulin Signaling

Analysis of Development Edited by Prof Benjamin H Willier, Prof Paul A Weiss and Prof Viktor Hamburger Pp xii + 735 (Philadelphia and London: W B Saunders Company, 1955) 105s

Mechanisms of Development

The LKB1 tumor suppressor gene, mutated in Peutz-Jeghers syndrome, encodes a serine/threonine kinase of unknown function. Here we show that mice with a targeted disruption of Lkb1 die at midgestation, with the embryos showing neural tube defects, mesenchymal cell death, and vascular abnormalities. Extraembryonic development was also severely affected; the mutant placentas exhibited defective labyrinth layer development and the fetal vessels failed to invade the placenta. These phenotypes were associated with tissue-specific deregulation of vascular endothelial growth factor (VEGF) expression, including a marked increase in the amount of VEGF messenger RNA. Moreover, VEGF production in cultured Lkb1(-/-) fibroblasts was elevated in both normoxic and hypoxic conditions. These findings place Lkb1 in the VEGF signaling pathway and suggest that the vascular defects accompanying Lkb1 loss are mediated at least in part by VEGF.

https://science.sciencemag.org/content/sci/293/5533/1323.full.pdf

Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice

Retarded Growth and Deficits in the Enteric and Parasympathetic Nervous System in Mice Lacking GFRα2, a Functional Neurturin Receptor

CLP-36 PDZ-LIM protein associates with nonmuscle alpha-actinin-1 and alpha-actinin-4.

Low-affinity neurotrophin receptor (LANR) and trk receptor tyrosine kinases (trks) serve as low- and high-affinity receptors for neurotrophins. Besides promoting the development and maintenance of the mammalian nervous system, it has been suggested that neurotrophins may have broader functions in the development of non-neuronal tissues. To evaluate the possible roles of neurotrophic factors in tooth development, we performed a detailed examination of the expression patterns of neurotrophin receptors during development of the rat tooth from initiation to completion of crown morphogenesis. mRNA expression was studied by in situ hybridisation and LANR protein was localised by immunohistochemistry. Furthermore, dissected tooth germs were cultured in vitro to examine the role of trigeminal innervation in the expression of neurotrophin receptors. mRNAs for LANR, trkB, and trkC, but not trkA, were detected in developing teeth. LANR and the truncated form of trkB, which lacks the intracellular tyrosine kinase domain, were expressed throughout tooth morphogenesis and their expression patterns were largely non-overlapping and changed spatio-temporally. trkC was expressed after birth, and it was restricted to dental papilla mesenchyme. The expression of all receptors correlated with the development of innervation, but, in addition, the expression of LANR and trkB appeared to be associated with cell differentiation and epithelial-mesenchymal interactions. The patterns of LANR, trkB, and trkC in teeth which underwent morphogenesis in organ culture were similar to those in vivo, which indicates that the expression of these neurotrophin receptors is not regulated by and does not depend on trigeminal innervation. The data suggest that neurotrophin receptors have roles in the development of tooth innervation, but that they also have non-neuronal, organogenetic functions. © 1996 Wiley-Liss, Inc.

https://anatomypubs.onlinelibrary.wiley.com/doi/pdf/10.1002/%28SICI%291097-0177%28199605%29206%3A1%3C87%3A%3AAID-AJA8%3E3.0.CO%3B2-X

Expression of neurotrophin receptors during rat tooth development is developmentally regulated, independent of innervation, and suggests functions in the regulation of morphogenesis and innervation.

To analyze the roles of neurotrophins during early development of rat teeth, we studied the expression of neurotrophin mRNAs from the initiation of first molar formation to the completion of crown morphogenesis. With RNAase protection assay all neurotrophin mRNAs were detected in embryonic teeth. In situ hybridization analysis revealed developmentally changing, distinct expression patterns for nerve growth factor (NGF) and neurotrophin-3 (NT-3), which were shown not to be regulated by or dependent on peripheral innervation. NGF mRNAs appeared in the mesenchymal target field of the tooth at the time of the trigeminal axon ingrowth (embryonic days 14-15: E14-E15), and they were also present along the pathway taken by growing trigeminal axons. NT-4/5 mRNAs were uniformly expressed in all epithelial cells, but brain-derived neurotrophic factor (BDNF) transcripts were not detected. All neurotrophins induced neurite outgrowth from E13-E16 trigeminal ganglion explants. These results suggest that NGF is involved in the guidance of trigeminal axons to embryonic teeth. In postnatal teeth, expression of NGF mRNAs, but not other neurotrophins, correlated with trigeminal axon ingrowth, proposing that NGF is involved in local sprouting and establishment of the final innervation pattern of the dental papilla and dentin. These results suggest that NGF is required for tooth innervation and that other neurotrophins may also have regulatory roles. In addition, the expression patterns of NGF, NT-3, and NT-4/5 as well as of neurotrophin receptors suggest that the neurotrophin system may also serve non-neuronal functions during tooth development.

Keijo Luukko

Papers

Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice

Retarded Growth and Deficits in the Enteric and Parasympathetic Nervous System in Mice Lacking GFRα2, a Functional Neurturin Receptor

CLP-36 PDZ-LIM protein associates with nonmuscle alpha-actinin-1 and alpha-actinin-4.

Expression of neurotrophin receptors during rat tooth development is developmentally regulated, independent of innervation, and suggests functions in the regulation of morphogenesis and innervation.

Neurotrophin mRNA expression in the developing tooth suggests multiple roles in innervation and organogenesis.