Excitotoxic loss of post-synaptic sites is distinct temporally and mechanistically from neuronal death.
Dendritic degeneration and loss of synaptic proteins are early events correlated with functional decline in neurodegenerative disease. The temporal and mechanistic relationship between synapse loss and cell death, however, remains unclear. We used confocal microscopy and image processing to count post-synaptic sites on rat hippocampal neurons by expressing post-synaptic density protein 95 fused to green fluorescent protein. Fluorescent puncta co-localized with neurotransmitter release sites, NMDA-induced Ca2+ increases and NMDA receptor immunoreactivity. During excitotoxic neurodegeneration, synaptic sites were lost and synaptic transmission impaired. These changes were mediated by NMDA receptors and required Ca2+-dependent activation of the proteasome pathway. Tracking synapses from the same cell following brief neurotoxic insult revealed transient loss followed by recovery. The time-course, concentration-dependence and mechanism for loss of post-synaptic sites were distinct from those leading to cell death. Cells expressing p14ARF, which inhibits ubiquitination of post-synaptic density protein 95 and prevents loss of synaptic sites, displayed an increased sensitivity to glutamate-induced cell death. Thus, excitotoxic synapse loss may be a disease-modifying process rather than an obligatory step leading to cell death. These results demonstrate the importance of assessing synaptic function independent of neuronal survival during neurodegeneration and indicate that this approach will be useful for identifying toxins that degrade synaptic connections and for screening for agents that protect synaptic function.
Waataja
Jonathan J
JJ
Department of Pharmacology and Graduate Program in Neuroscience, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Kim
Hee Jung
HJ
Roloff
Alan M
AM
Thayer
Stanley A
SA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D000076122:Disks Large Homolog 4 Protein; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D005786:Gene Expression Regulation / Q000187:drug effects; D018698:Glutamic Acid / Q000633:toxicity*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D007004:Hypoglycemic Agents; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects*; D009498:Neurotoxins / Q000633:toxicity*; D010168:Palmitates / Q000494:pharmacology; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D013569:Synapses / Q000187:drug effects* / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection / Q000379:methods
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009498:Neurotoxins; D010168:Palmitates; D016194:Receptors, N-Methyl-D-Aspartate; D049452:Green Fluorescent Proteins; C022776:2-bromopalmitate; D018698:Glutamic Acid; D002118:Calcium