ABSTRACT
Recent studies in mice have clearly demonstrated that eliminating Apo E alters the rate, character and distribution of A beta deposits. In the present study, we asked whether elevating the levels of Apo E can, in a dominant fashion, influence amyloid deposition. We expressed human (Hu) Apo E4 via the mouse prion protein promoter, resulting in high expression in both astrocytes and neurons; only astrocytes efficiently secreted Hu Apo E4 (at least 5-fold more than endogenous). Mice hyper-expressing Hu Apo E4 developed normally and lived normal lifespans. The co-expression of Hu Apo E4 with a mutant amyloid precursor protein (APP) (Mo/Hu APPswe) or mutant APP and mutant presenilin (PS1dE9) did not lead to proportional changes in the age of appearance, relative burden, character or distribution of A beta deposits. We suggest that these data are best explained by proposing that the mechanisms by which Apo E influences A beta deposition involves an aspect of its normal function that is not augmented by hyper-expression.
Subject(s)
Apolipoproteins E/metabolism , Astrocytes/metabolism , Neurons/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Apolipoprotein E4 , Apolipoproteins E/genetics , Astrocytes/cytology , Brain/metabolism , Cells, Cultured , Gene Expression Regulation , Humans , Immunoblotting , Mice , Mice, Transgenic , Mutation , Neurons/cytology , Time FactorsABSTRACT
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
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.
