Title

Isolation of a brain fraction enriched in GABAergic type-II postsynaptic densities. Characterization of the GABAA receptors and septin 11, two proteins enriched in this fraction

Date of Completion

January 2008

Keywords

Biology, Neuroscience

Degree

Ph.D.

Abstract

The γ-Aminobutyric acid type A receptors (GABAARs) concentrate at the postsynaptic membrane of GABAergic synapses. I have devised a method for the purification of a brain fraction enriched in type-II GABAergic postsynaptic densities (PSDs) that is enriched in GABAARs and gephyrin. I have separated this fraction from another fraction enriched in glutamatergic type-I PSDs and from a fraction enriched in lipid rafts. This separation was accomplished by subjecting the Triton X-100 insoluble "One Triton PSD" fraction to ultracentrifugation in a sucrose gradient due to the different buoyant densities of these fractions. The type-II PSD fraction contained GABAARs with subunit composition corresponding to that of the synaptic GABAARs. The lower density fraction of detergent resistant domains (DRMs) enriched in lipid rafts contained GABAARs with a subunit composition corresponding to that of the non-synaptic GABAARs. I also found two pools of GABAARs insoluble in Triton X-100 at 4°C in cultured hippocampal neurons: a pool of the synaptic clustered GABAARs and a pool of non-synaptic diffusely distributed GABAARs, the latter associated to lipid rafts. These two pools had different subunit composition. Thus synaptic GABAARs associated with type-II PSDs had subunit composition and physical properties different from those of the extrasynaptic GABAARs involved in tonic inhibition. ^ Septin 11 was identified as the main component of the main protein band enriched in type-II PSD over type-I PSD. Septin 11 was highly expressed in the molecular layer of the cerebellum and olfactory bulb. Many GABAergic synapses but fewer glutamatergic synapses have associated septin 11 clusters in cultured hippocampal neurons and intact rat brain sections. Moreover septin 11 clusters concentrated at the base of dendritic spines, and at the bifurcation points of dendritic branches. Knocking down septin 11 with septin 11 shRNAs and overexpressing dominate-negative EGFP—or mCherry—septin 11 fusion proteins in cultured hippocampal neurons led to (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic spines; and (iii) decreased GABAergic synaptic contacts that these neurons received. Overexpression of non-tagged septin 11 in cultured hippocampal neurons did not show significant effect on the cell morphology. The results indicate that septin 11 concentrates at some GABAergic synapses and plays an important role in the cytoarchitecture of the dendritic branches and dendrite spines. ^