ABSTRACT
Although functional neuromuscular junctions (NMJs) form in NCAM-deficient mice, they exhibit multiple alterations in presynaptic organization and function. Profound depression and unusual periodic total transmission failures with repetitive stimulation point to a defect in vesicle mobilization/cycling, and these defects were mimicked in (+/+) NMJs by inhibitors of myosin light chain kinase, known to affect vesicle mobilization. Two separate release mechanisms, utilizing different endocytic machinery and Ca(2+) channels, were shown to coexist in (-/-) terminals, with the mature process targeted to presynaptic membrane opposed to muscle, and an abnormally retained immature process targeted to the remainder of the presynaptic terminal and axon. Thus, NCAM plays a critical and heretofore unsuspected role in the molecular organization of the presynaptic NMJ.
Subject(s)
Neural Cell Adhesion Molecules/deficiency , Neural Cell Adhesion Molecules/genetics , Neuromuscular Junction/metabolism , Neurotransmitter Agents/metabolism , Synaptic Transmission/genetics , Synaptic Vesicles/metabolism , Animals , Calcium Channels/genetics , Calcium Channels/metabolism , Electric Stimulation , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Cell Adhesion Molecules/metabolism , Neuromuscular Junction/genetics , Neurotransmitter Agents/genetics , Presynaptic Terminals/metabolism , Synaptic Vesicles/geneticsABSTRACT
Most synapses contain high concentrations of neurotransmitter receptors in the postsynaptic plasma membrane. Agrin (Ag) is an extracellular matrix protein necessary for the localization of acetylcholine receptors at the neuromuscular junction and for the differentiation of synapses in hippocampal neurons in vitro. The temporal pattern of agrin expression during the development of the central nervous system (CNS) is consistent with the notion that agrin expression is regulated during synaptogenesis. To identify the processes underlying this regulation, we have analyzed levels and alternative splicing of agrin mRNA in primary hippocampal neurons. Our results indicate that in the initial phases of synapse formation, contact-mediated processes and action potential-dependent neurotransmission regulate agrin mRNA expression, while secreted factors from glial cells, but not from hippocampal neurons, influence the alternative splicing of agrin mRNA. Previous studies have shown that specific agrin isoforms are able to induce the activation of a transcription factor and that secreted agrin associates with cellular surfaces. Therefore, we have tested whether agrin isoforms contribute to the contact-mediated induction of agrin expression in hippocampal neurons. None of the agrin isoforms tested in this study revealed this activity. Finally, we show that the role of evoked neural transmission in controlling agrin transcription changes during differentiation in vitro.
Subject(s)
Action Potentials/physiology , Agrin/genetics , Alternative Splicing , Cell Communication/physiology , Gene Expression Regulation/physiology , Hippocampus/physiology , Neurons/physiology , Synapses/physiology , Animals , Cells, Cultured , Embryo, Mammalian , Hippocampus/cytology , Kinetics , Mice , Mice, Inbred C57BL , Neurons/cytology , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
Agrin controls the formation of the neuromuscular junction. Whether it regulates the differentiation of other types of synapses remains unclear. Therefore, we have studied the role of agrin in cultured hippocampal neurons. Synaptogenesis was severely compromised when agrin expression or function was suppressed by antisense oligonucleotides and specific antibodies. The effects of antisense oligonucleotides were found to be highly specific because they were reversed by adding recombinant agrin and could not be detected in cultures from agrin-deficient animals. Interestingly, the few synapses formed in reduced agrin conditions displayed diminished vesicular turnover, despite a normal appearance at the EM level. Thus, our results demonstrate the necessity of agrin for synaptogenesis in hippocampal neurons.
Subject(s)
Agrin/metabolism , Cell Differentiation/physiology , Hippocampus/metabolism , Neurons/metabolism , Synapses/metabolism , Agrin/genetics , Agrin/pharmacology , Animals , Antibodies/pharmacology , Cell Count , Cell Differentiation/drug effects , Cells, Cultured , Electric Stimulation , Endocytosis/drug effects , Evoked Potentials/drug effects , Exocytosis/drug effects , Hippocampus/cytology , Hippocampus/drug effects , Neurons/cytology , Neurons/drug effects , Oligonucleotides, Antisense/pharmacology , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Synapses/ultrastructure , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Tetrodotoxin/pharmacologyABSTRACT
The synaptic basal lamina protein agrin is essential for the formation of neuromuscular junctions. Agrin mediates the postsynaptic clustering of acetylcholine receptors and regulates transcription in muscles. Agrin expression is not restricted to motor neurons but can be demonstrated throughout the CNS. The functional significance of agrin expression in neurons other than motor neurons is unknown. To test whether agrin triggers responses in neurons that lead to the activation of transcription factors, we have analyzed phosphorylation of the transcriptional regulatory site serine 133 of the transcription factor CREB (cAMP response element binding protein) in primary hippocampal neurons. Our results indicate that the neuronal (Ag4,8), but not the non-neuronal (Ag0,0), isoform of agrin induces CREB phosphorylation in hippocampal neurons. The kinetics of agrin- and BDNF-induced CREB phosphorylation are similar: peak levels are reached in minutes and are strongly reduced 2 hr later. Neuronal responses to agrin require extracellular calcium, and, in contrast to tyrosine kinase inhibitors, the specific inhibition of protein kinase A (PKA) does not affect agrin-evoked CREB phosphorylation. Our results show that hippocampal neurons specifically respond to neuronal agrin in a Ca2+-dependent manner and via the activation of tyrosine kinases.
