Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents in trkB (Ϫ/Ϫ) and trkC (Ϫ/Ϫ) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10-P16) did not reveal major differences in the topographic patterns of hippocampal connections.In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21-49%) and decreased densities of axonal varicosities (8-17%) in both trkB (Ϫ/Ϫ) and trkC (Ϫ/Ϫ) mice. In addition, electron microscopic analyses showed that trkB (Ϫ/Ϫ) and trkC (Ϫ/Ϫ) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially in trkB (Ϫ/Ϫ) mice. We conclude that neither trkB nor trkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNS in vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.
Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A 2 (cPLA 2 ) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA 2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4-dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.
Dystrophic neurites are major components of neuritic (both immature and mature) senile plaques in Alzheimer disease. Previous studies have shown strong immunoreactivity for different neuropeptides, and chromogranin A, a protein associated with dense-core vesicles, in dystrophic neurites. In the present study, antibodies to synaptophysin, synapsin, Rab3a and synaptotagmin (synaptic vesicle proteins), and SNAP-25 (synaptosomal-associated protein of 25 kD) and syntaxin (presynaptic plasma membrane proteins) have been used to learn about the dystrophic neurite equipment of proteins that are necessary for the docking and fusion of synaptic vesicles, and then for exocytosis and neurotransmission. The present results have shown that, although most neuritic senile plaques have chromogranin A- and SNAP-25-immunoreactive dystrophic neurites, only a percentage of them contain synaptophysin, and a minority contain synaptotagmin and Rab3a. Dystrophic neurites do not contain synapsin and syntaxin. These results show that dystrophic neurites of senile plaques are defective in proteins that control exocytosis and neurotransmission.
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