Even though many extracellular factors have been identified as promoters of general dendritic growth and branching, little is known about the cell-intrinsic modulators that allow neurons to sculpt distinctive patterns of dendrite arborization. Here, we identify Lrig1, a nervous system-enriched LRR protein, as a key physiological regulator of dendrite complexity of hippocampal pyramidal neurons. Lrig1-deficient mice display morphological changes in proximal dendrite arborization and defects in social interaction. Specifically, knockdown of Lrig1 enhances both primary dendrite formation and proximal dendritic branching of hippocampal neurons, two phenotypes that resemble the effect of BDNF on these neurons. In addition, we show that Lrig1 physically interacts with TrkB and attenuates BDNF signaling. Gain and loss of function assays indicate that Lrig1 restricts BDNF-induced dendrite morphology. Together, our findings reveal a novel and essential role of Lrig1 in regulating morphogenic events that shape the hippocampal circuits and establish that the assembly of TrkB with Lrig1 represents a key mechanism for understanding how specific neuronal populations expand the repertoire of responses to BDNF during brain development.
The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.
1 The present study attempted to pharmacologically characterize the muscarinic receptor subtypes mediating contraction of human umbilical vein (HUV). 2 HUV rings were mounted in organ baths and concentration-response curves were constructed for acetylcholine (ACh) (pEC 50 : 6.1670.04; maximum response 80.0071.98% of the responses induced by serotonin 10 mM). The absence of endothelium did not modify the contractile responses of ACh in this tissue. 3 The role of cholinesterases was evaluated: neither neostigmine (acetylcholinesterase inhibitor) nor iso-OMPA (butyrylcholinesterase inhibitor) modified ACh responses. When both enzymes were simultaneously inhibited, a significantly but little potentiation was observed (control: pEC 50 6.3370.03; double inhibition: pEC 50 6.5770.05). 4 Atropine, nonselective muscarinic receptors antagonist, inhibited ACh-induced contraction (pK B 9.67). The muscarinic receptors antagonists pirenzepine (M 1 ), methoctramine (M 2 ) and pFHHSiD (M 3 ) also antagonized responses to ACh. The affinity values estimated for these antagonists against responses evoked by ACh were 7.58, 6.78 and 7.94, respectively. On the other hand, PD 102807 (M 4 selective muscarinic receptors antagonist) was ineffective against ACh-induced contraction. 5 In presence of a blocking concentration of pirenzepine, pFHHSiFD produced an additional antagonism activity on ACh-induced responses. 6 The M 1 muscarinic receptors agonist McN-A-343 produced similar maximum but less potent responses than ACh in HUV. The calculated pA 2 for pirenzepine against McN-A-343 induced responses was 8.54. 7 In conclusion, the data obtained in this study demonstrate the role of M 1 muscarinic receptor subtypes and suggest the involvement of M 3 muscarinic receptor subtypes in ACh-induced vasoconstriction in HUV rings. In addition, the vasomotor activity evoked by ACh does not seem to be modulated by endothelial factors, and their enzymatic degradation appears to have little functional relevance in this tissue.
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