ABSTRACT
The development of novel therapeutic strategies for Alzheimer's disease (AD) represents one of the biggest unmet medical needs today. Application of neurotrophic factors able to modulate neuronal survival and synaptic connectivity is a promising therapeutic approach for AD. We aimed to determine whether the loco-regional delivery of ciliary neurotrophic factor (CNTF) could prevent amyloid-beta (Abeta) oligomer-induced synaptic damages and associated cognitive impairments that typify AD. To ensure long-term administration of CNTF in the brain, we used recombinant cells secreting CNTF encapsulated in alginate polymers. The implantation of these bioreactors in the brain of Abeta oligomer-infused mice led to a continuous secretion of recombinant CNTF and was associated with the robust improvement of cognitive performances. Most importantly, CNTF led to full recovery of cognitive functions associated with the stabilization of synaptic protein levels in the Tg2576 AD mouse model. In vitro as well as in vivo, CNTF activated a Janus kinase/signal transducer and activator of transcription-mediated survival pathway that prevented synaptic and neuronal degeneration. These preclinical studies suggest that CNTF and/or CNTF receptor-associated pathways may have AD-modifying activity through protection against progressive Abeta-related memory deficits. Our data also encourage additional exploration of ex vivo gene transfer for the prevention and/or treatment of AD.
Subject(s)
Alzheimer Disease/complications , Ciliary Neurotrophic Factor/biosynthesis , Ciliary Neurotrophic Factor/therapeutic use , Memory Disorders/etiology , Memory Disorders/therapy , Synapses/drug effects , Alzheimer Disease/genetics , Amyloid beta-Peptides/administration & dosage , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/pharmacology , Amyloid beta-Protein Precursor/genetics , Animals , Apoptosis/genetics , Brain/pathology , Cell Count/methods , Cell- and Tissue-Based Therapy/methods , Cells, Cultured , Ciliary Neurotrophic Factor/administration & dosage , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Green Fluorescent Proteins/genetics , Humans , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation/genetics , Neurons/drug effects , Neurons/metabolism , Peptide Fragments/administration & dosage , Peptide Fragments/pharmacology , Signal Transduction/drug effects , Synapses/metabolism , Synaptosomes/metabolism , Synaptosomes/pathology , Synaptosomes/ultrastructure , Time Factors , Transfection/methodsABSTRACT
NMDA receptors (NMDARs) are essential for modulating synaptic strength at central synapses. At hippocampal CA3-to-CA1 synapses of adult mice, different NMDAR subtypes with distinct functionality assemble from NR1 with NR2A and/or NR2B subunits. Here we investigated the role of these NMDA receptor subtypes in long-term potentiation (LTP) induction. Because of the higher NR2B contribution in the young hippocampus, LTP of extracellular field potentials could be enhanced by repeated tetanic stimulation in young but not in adult mice. Similarly, NR2B-specific antagonists reduced LTP in young but only marginally in adult wild-type mice, further demonstrating that in mature CA3-to-CA1 connections LTP induction results primarily from NR2A-type signaling. This finding is also supported by gene-targeted mutant mice expressing C-terminally truncated NR2A subunits, which participate in synaptic NMDAR channel formation and Ca2+ signaling, as indicated by immunopurified synaptic receptors, postembedding immunogold labeling, and spinous Ca2+ transients in the presence of NR2B blockers. These blockers abolished LTP in the mutant at all ages, revealing that, without the intracellular C-terminal domain, NR2A-type receptors are deficient in LTP signaling. Without NR2B blockade, CA3-to-CA1 LTP was more strongly reduced in adult than young mutant mice but could be restored to wild-type levels by repeated tetanic stimulation. Thus, besides NMDA receptor-mediated Ca2+ influx, subtype-specific signaling is critical for LTP induction, with the intracellular C-terminal domain of the NR2 subunits directing signaling pathways with an age-dependent preference.
Subject(s)
Long-Term Potentiation/physiology , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Brain Chemistry , Calcium Signaling/drug effects , Calcium Signaling/physiology , Electric Stimulation , Excitatory Amino Acid Antagonists/pharmacology , Gene Targeting , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/physiology , In Vitro Techniques , Mice , Mice, Mutant Strains , Protein Structure, Tertiary/physiology , Protein Subunits/antagonists & inhibitors , Protein Subunits/chemistry , Protein Subunits/metabolism , Pyramidal Cells/drug effects , Pyramidal Cells/metabolism , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/genetics , Subcellular Fractions/chemistry , Synapses/metabolism , Synapses/ultrastructureABSTRACT
Glutamate neurotransmission in the olfactory bulb involves both axodendritic synapses and dendrodendritic reciprocal synapses and possibly also extrasynaptic receptors. By using a sensitive immunogold procedure, we have investigated the organization of two synaptic scaffolding molecules, PSD-95 and PSD-93, as well as N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors, at these heterogeneous glutamate signaling sites. Immunolabeling for PSD-95 and PSD-93 was present in all major types of putative glutamatergic synapse, suggesting that these proteins are essential components of the synaptic signaling apparatus. The linear density and the subsynaptic distribution of PSD-95/PSD-93 gold particles did not differ significantly between axodendritic and dendrodendritic synapses. Antibodies recognizing NMDA and AMPA receptor subunits also labeled asymmetric synapses throughout the olfactory bulb. Immunolabeling for the AMPA receptor subunits GluR2/3 was similar in all types of synapse. In contrast, immunogold signals for the NR1 subunit of NMDA receptors varied significantly among different synapse populations, with olfactory nerve synapses in the glomerular layer showing the lowest labeling intensity. Although the lateral dendrites of mitral and tufted cells have been reported to respond to glutamate, they did not display significant plasma membrane labeling for the NR1 subunit or for PSD-95, suggesting that the physiological effects of glutamate at these sites are mediated by NMDA autoreceptors that are not clustered and occur only at a low density on the dendritic surface. Our quantitative analysis of olfactory bulb synapses indicates that the density of NMDA receptors is not determined by the complement of PSD-95/PSD-93. The latter molecules appear to be expressed in an all-or-none fashion and may form a standard lattice common to different types of glutamatergic synapse.
