Dynamic Structural Properties of Synapses

National Institute Of Neurological Disorders And Stroke

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Abstract

Past years focus was on two proteins, densin and FAM81A, both found to be highly enriched in affinity-purified PSD-95 complexes. Immuno-gold labeling of cultured hippocampal neurons shows concentration of densin at PSDs, but also some labeling at extrasynaptic plasma membranes of soma and dendrites and endoplasmic reticulum. At the PSD, the median distance of label from the postsynaptic membrane was 27 nm, with the majority of label (90 %) confined within 40 nm from the postsynaptic membrane, indicating predominant localization of densin at the PSD core. Depolarization (treatment with high K+ for 2 min), a condition previously shown to promote major re-organization of PSD components, caused only a slight shift in the median distance of densin label, without any significant change in the overall label density at the PSD. We hypothesize that, under excitatory conditions, the relatively stable densin molecules firmly embedded within the PSD would anchor and target activated CaMKII to its substrates, SynGAP and AIDA-1, located at the PSD core. The product of the gene FAM81A, is a protein of unknown function and, so far, no published articles exist that focus on the gene and/or protein. FAM81A mRNA and protein expression are highest in brain tissue (Human Protein Atlas) and our mass spectrometric analysis of isolated PSDs indicated a stoichiometry of one FAM81A to fourteen PSD-95 (Lowenthal et al 2015). Past years experiments showed that FAM81A protein in brain is expressed late in development, with a post-natal gradual increase in levels that parallels the expression of PSD-95. Comparison of subcellular fractions from adult brain by western immunoblotting indicated a distribution of FAM81A similar to PSD-95, with a drastic enrichment in the PSD fraction. Immuno-electron microscopy on adult brain revealed specific immunogold labeling for FAM81A at a subpopulation of PSDs. The label for FAM81A was concentrated at the outer edge of the PSD core, within 30-40 nm from the postsynaptic membrane. On the other hand, an NCBI Conserved Domain search revealed a sequence on FAM81A similar to the SMC_N (N-terminus of Structural Maintenance of Chromosomes) domain, hinting at an interaction of the protein with chromosomes. Also, preliminary results from our commissioned yeast two hybrid study suggested two nuclear binding partners for FAM81A (polyadenylate-binding protein 2 and mediator of RNA polymerase II transcription subunit 10). In conclusion, while biochemical and EM evidence establish FAM81A as a genuine component of the PSD, sequence analysis and Y2H studies, hint at a role for the protein in the nucleus. Further studies will focus on a possible function of FAM81A as a signaling molecule between synapse and nucleus.